Carcinogenicity and Mutagenicity of Nanoparticles - Biozid

Transcription

TEXTE
50/2014
Carcinogenicity
and Mutagenicity of
Nanoparticles –
Assessment of Current
Knowledge as Basis for
Regulation
TEXTE 50/2014
Environmental Research of the
Federal Ministry for the
Environment, Nature Conservation,
Building and Nuclear Safety
Project No. (FKZ) 3709 61 220
Report No. (UBA-FB) 001725/E
Carcinogenicity and Mutagenicity of
Nanoparticles – Assessment of Current
Knowledge as Basis for Regulation
by
Katrin Schröder
Christina Pohlenz-Michel
Nelly Simetska
Jens-Uwe Voss
Sylvia Escher
Inge Mangelsdorf
Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover
On behalf of the Federal Environment Agency (Germany)
Imprint
Publisher:
Umweltbundesamt
Wörlitzer Platz 1
06844 Dessau-Roßlau
Tel: +49 340-2103-0
Fax: +49 340-2103-2285
[email protected]
Internet: www.umweltbundesamt.de
/umweltbundesamt.de
/umweltbundesamt
Study performed by:
Fraunhofer Institute for Toxicology and Experimental Medicine
Nikolai-Fuchs-Str. 1, 30625 Hannover
Study completed in:
September 2013
Edited by:
Section II 1.2 Toxikologie, Gesundheitsbezogene Umweltbeobachtung
Petra Apel
Publication as pdf:
http://www.umweltbundesamt.de/publikationen/carcinogenicity-mutagenicity-of-nanoparticles
ISSN 1862-4804 DessauRoßlau, July 2014
The Project underlying this report was supported with funding from the Federal
Ministry for the Environment, Nature Conservation, Building and Nuclear safety
under project number FKZ 3709 61 220. The responsibility for the content of this
publication lies with the author(s).
Abstract
Carcinogenicity studies with several types of respirable particles and fibres indicate a carcinogenic potential
from inhalation and there is concern that the carcinogenic potency of nanomaterials is higher than for the
corresponding micromaterials. In this research project, long term studies with nanomaterials were used to
identify relevant indicators of toxicity of nanomaterials including possible precursors of carcinogenicity. Due
to the heterogeneous characteristics of the materials and the different study types, a structured and systematic
data analysis was performed by means of a relational database (PaFtox). More than 100 inhalation studies
and instillation studies with rodents with Carbon Black, silicon dioxide, metals or metal oxides, and carbonnanotubes were analysed. Effects like neutrophil number, total protein and LDH content in the bronchioalveolar lavage fluid (BALF) are frequently measured and are sensitive indicators of toxicity of all particles
investigated. In addition, infiltration of inflammatory cells in the lung and increased lung weights are often
observed. The LOELs of nano-objects are generally lower than the LOELs of the corresponding larger
objects and they differ by several orders of magnitude between analysed substances: Silver was identified as
the most toxic nanomaterial within our selection of nanomaterials. Sustained inflammation can be seen as
one possible early event in the sequence of cancer development and nanomaterials can be grouped on basis
of their potential to generate inflammation. A preliminary LOEL (based on inflammatory parameters) of
0.1 mg/m3 (exposure 24 h/d, 7 d/wk) is proposed to distinguish the so called “inert” nanomaterials (e.g.
Carbon Black) from nanomaterials with specific toxicity. Our data further support to have nanotubes in a
separate group.
Kurzbeschreibung
Kanzerogenitätsstudien mit verschiedenen alveolengängigen Partikeln und Fasern deuten auf ein
kanzerogenes Potential bei inhalativer Exposition hin und es wird befürchtet, dass dieses Potential bei
Nanomaterialen höher ist als beim entsprechenden Mikromaterial. In diesem Forschungsprojekt wurden
Langzeitstudien mit Nanomaterialien analysiert, um relevante Indikatoren der Toxizität einschließlich
möglicher Vorstufen der Kanzerogenität von Nanomaterialien zu identifizieren. Eine strukturiertere und
systematische Analyse der heterogenen Materialeigenschaften und der unterschiedlichen Studientypen wurde
mit Hilfe einer relationalen Datenbank durchgeführt. Mehr als 100 Inhalations- und Instillationsstudien mit
Carbon Black, Siliziumdioxid, Metallen oder Metalloxiden an Nagern wurden analysiert. Häufig werden
Effekte wie Neutrophilen-Anzahl, Gesamtprotein- und LDH-Gehalt in der bronchio-alveolären
Lavageflüssigkeit (BALF) gemessen, sie sind sensitive Indikatoren für die Toxizität der untersuchten
Partikel. Zudem werden oft Infiltration von Entzündungszellen in der Lunge und erhöhtes Lungengewicht
beobachtet. Die LOELs der Nano-Objekte sind tendenziell niedriger als die LOELs der entsprechenden
größeren Objekte und sie unterscheiden sich durch mehrere Größenordnungen: In unserer Auswahl an
Nanomaterialien hatte Silber das höchste toxische Potential. Chronische Entzündung kann als möglicher
früher Vorläufer der Krebsentstehung betrachtet werden, und Nanomaterialien können anhand ihres
Potentials Entzündungen zu verursachen gruppiert werden. Ein vorläufiger LOEL (basierend auf
Entzündungsparametern) von 0.1 mg/m3 (Exposition 24 h/d, 7 d/w) wird vorgeschlagen, um die sogenannten
“inerten” Nanomaterialien (z.B. Carbon Black) von Nanomaterialien mit spezifischer Toxizität zu
unterscheiden. Unsere Daten unterstreichen den Ansatz, dass Nanotubes in eine separate Gruppe gehören.
Table of Contents
List of Figures
List of Tables
Abbreviations
1
2
Introduction.............................................................................................................................................. 1
1.1
Hazard and risk of nano-objects ....................................................................................................... 1
1.2
Nanomaterials –Definitions.............................................................................................................. 2
Methods ................................................................................................................................................... 3
2.1
Literature search ............................................................................................................................... 3
2.1.1 Approach..................................................................................................................................... 3
2.1.2 Results......................................................................................................................................... 4
2.2
Data analysis by means of a relational database .............................................................................. 5
2.2.1 Database Structure ...................................................................................................................... 5
2.2.2 Particle and fibre characterisation............................................................................................... 7
2.2.3 Selection criteria for studies and materials ................................................................................. 7
2.2.4 Study design................................................................................................................................ 8
2.2.5 Toxicological data ...................................................................................................................... 9
2.2.6 Quality assurance ........................................................................................................................ 9
2.2.7 Database queries ....................................................................................................................... 12
3
Data analysis .......................................................................................................................................... 13
3.1
Particle and fibre characterisation .................................................................................................. 13
3.1.1 Specification of primary particles and fibres by producer / supplier (as produced) or
by authors (as received) ........................................................................................................... 13
3.1.2 Specification of secondary particles and fibres by authors (as administered) .......................... 15
3.2
Studies included ............................................................................................................................. 16
3.2.1 Routes and study durations ....................................................................................................... 16
3.2.2 Species ...................................................................................................................................... 19
3.2.3 Dose levels, NOELs and LOELs .............................................................................................. 19
3.3
Particles included ........................................................................................................................... 20
3.4
Parameter / Effect analysis ............................................................................................................. 21
3.4.1 Target / organs and effects ........................................................................................................ 21
3.4.2 Reversibility of effects .............................................................................................................. 25
3.4.3 Parameters affecting toxicity of particles and fibres................................................................. 28
I
3.4.3.1 Composition.............................................................................................................................. 29
3.4.3.2 Tubes versus particles ............................................................................................................... 34
3.4.3.3 Diameter and specific Surface .................................................................................................. 35
3.4.4 Genotoxicity of nano-objects in vivo........................................................................................ 37
3.4.5 Carcinogenicity ......................................................................................................................... 39
3.5
Correlations .................................................................................................................................... 41
3.5.1 Cell counts ................................................................................................................................ 41
3.5.2 Alveolar macrophages .............................................................................................................. 46
3.5.3 Total Protein and lactate dehydrogenase .................................................................................. 46
3.5.4 Infiltration ................................................................................................................................. 50
3.5.5 Lung weight .............................................................................................................................. 51
4
Discussion .............................................................................................................................................. 54
4.1
Results ............................................................................................................................................ 54
4.1.1 Carcinogenicity ......................................................................................................................... 54
4.1.2 Availability of studies for different particles and fibres ........................................................... 54
4.1.3 Particle and fibre characterisation............................................................................................. 55
4.1.4 Application routes ..................................................................................................................... 56
4.1.5 Dose measure ............................................................................................................................ 58
4.1.6 Target organs and important effects ......................................................................................... 59
4.1.7 New parameters ........................................................................................................................ 59
4.1.8 LOELs....................................................................................................................................... 60
4.1.9 Exposure duration versus time point ........................................................................................ 60
4.1.10 Overload ................................................................................................................................... 61
4.1.11 Reversibility of effects .............................................................................................................. 61
4.1.12 Genotoxicity ............................................................................................................................. 61
4.2
Concepts for Grouping of nanomaterials ....................................................................................... 61
4.2.1 Groups proposed in the literature.............................................................................................. 61
4.2.2 Grouping of nanomaterials for optimized testing strategies ..................................................... 64
5
Conclusion and outlook ......................................................................................................................... 67
6
References.............................................................................................................................................. 69
7
Appendix................................................................................................................................................ 72
7.1
Publications in PaFtox database (October 2012) ........................................................................... 72
7.2
Data analysis by databases ............................................................................................................. 75
7.2.1 NanoSafety Cluster ................................................................................................................... 75
II
7.2.2 DaNa ......................................................................................................................................... 75
7.2.3 Database on Research into the Safety of Manufactured Nanomaterials ................................... 75
7.2.4 Special features of the PaFtox database .................................................................................... 77
7.3
Study reports from PaFtox database (October 2012) ..................................................................... 77
III
List of figures
Fig 1:
Taxonomy of Nanomaterial; adapted from ISO/TR 12802:2010 & ISO/TS 80004-4:2011 ............. 3
Fig 2:
Data entry mask of the PaFtox database ........................................................................................... 6
Fig 3:
MMAD in inhalation studies (left) and hydrodynamic diameter in instillation studies
(right) ............................................................................................................................................. 16
Fig 4:
Overview effected targets / organs .................................................................................................. 22
Fig 5:
Number of effects per category time point in whole body inhalation studies on
carcinogenicity (query counted effects at minimum dose and earliest time point) ........................ 40
Fig 6:
Effect – “LOELs” per particle in whole body inhalation studies on carcinogenicity (query
counted effects at min dose and first time point) ........................................................................... 41
Fig 7:
Correlation of relative number of neutrophils with particle mass or particle surface area ............. 45
Fig 8:
Correlation of total protein with particle surface area..................................................................... 47
Fig 9:
Correlation of LDH with particle surface area ................................................................................ 49
Fig 10: Predicted fractional deposition of inhaled particles (Oberdörster et al., 2005) ............................... 56
Fig 11: Illustration of grouping of nanoparticles based on material properties and/or biological
effects (from Oomen et al. (2013)). ............................................................................................... 67
IV
List of Tables
Tab. 1: Concept of literature search .............................................................................................................. 4
Tab. 2: Examples for effects in BALF and lung ......................................................................................... 11
Tab. 3: Primary characterisation in studies with nano-objects .................................................................... 13
Tab. 4: Available information for reference material “Min-U-Sil 5”.......................................................... 14
Tab. 5: Secondary characterisation in studies with nano-objects ................................................................ 15
Tab. 6: Study design of inhalation studies................................................................................................... 17
Tab. 7: Study design of instillation studies ................................................................................................. 18
Tab. 8: Types of studies in rats and mice ..................................................................................................... 19
Tab. 9: Number of different dose levels ...................................................................................................... 20
Tab. 10:Particles included ............................................................................................................................ 20
Tab. 11:Number of studies for different substances and particle diameter categories and
application routes ........................................................................................................................... 21
Tab. 12:Observed effects .............................................................................................................................. 23
Tab. 13:Parameter / Effects beyond the respiratory tract ............................................................................. 24
Tab. 14:Parameter / Effect of study evaluation (+) or (-) postexposure in whole body inhalation
studies ............................................................................................................................................ 26
Tab. 15:Parameter / Effects (number) appearing only in the postexposure period at LOEL per time
point in studies with whole body exposure. ................................................................................... 28
Tab. 16:LOELs per route, substance and time point .................................................................................... 29
Tab. 17:Parameter / Effects occurring only with heavy metals and oxides.................................................. 30
Tab. 18:Parameter / Effects occurring only with silicon dioxides ............................................................... 33
Tab. 19:Parameter / Effects occurring only with carbon nanotubes............................................................. 34
Tab. 20:Influence of particle diameter on LOELs........................................................................................ 36
Tab. 21:Influence of specific surface on LOELs.......................................................................................... 37
Tab. 22:Genotoxic effects (measured and percentage positive effects) ....................................................... 38
Tab. 23:“LOELS” for positive genotoxic effects ......................................................................................... 39
Tab. 24:Investigation of cells in different targets (statistically significant increased/measured or
investigated) ................................................................................................................................... 42
Tab. 25:Frequency of increased / measured cell counts in BALF per time point category ......................... 43
Tab. 26:Time related distribution of measurements of total protein and lactate dehydrogenase ................. 46
Tab. 27:Number of studies positive infiltrations .......................................................................................... 50
Tab. 28:“LOELs” for infiltration and macrophage infiltration .................................................................... 51
Tab. 29:Minimum and Maximum percentages of burden of the lung weight .............................................. 52
Tab. 30:Minimum and maximum of percentage change of lung weight...................................................... 53
V
Tab. 31:“LOELs” for lung weight ................................................................................................................ 54
Tab. 32:Comparison of application routes ................................................................................................... 58
VI
List of Abbreviations
BALF
Bronchioalveolar lavage fluid
LDH
Lactate dehydrogenase
LOEL
Lowest observed effect level
MMAD
Mass median aerodynamic diameter
NOEL
No observed effect level
VSSA
Volume-specific surface area
PaFtox
Particle and Fibre toxicity
VII
1
1.1
Introduction
Hazard and risk of nano-objects
The general population is exposed to nanoparticles mainly in larger cities in form of fine dust. Workers can
be additionally exposed during the production and processing of nano-objects. Furthermore, the number of
consumer products based on nanotechnology is raising (e.g. shoe sprays).
Nano-objects differ regarding their physico-chemical properties (e.g. size, specific surface, zeta potential)
from larger particles or fibres, but cause similar effects (e.g. inflammation in the lung or other toxic effects
including cancer). However, based on the same particle mass, they are more toxic, among other reasons due
to their larger particle surface (Oberdörster et al., 2005). Therefore there is concern for higher carcinogenic
potency of nano-objects.
The data basis concerning carcinogenicity of nano-objects is very limited with respect to inhalation studies.
Also intratracheal studies as surrogate for inhalation studies, are limited. To our knowledge nanoparticles
and fine particles were carcinogenic in all existing inhalation and intratracheal studies with exposure
durations longer than 2 years (Roller, 2009). Most of them caused tumours at all concentrations tested.
However, even at the lowest dose levels often unrealistic high dose levels were used that overwhelm the
defence mechanisms in the lung (overload). Similarly, in vitro studies on genotoxicity or ROS-generation are
virtually all positive, in many cases at cytotoxic concentrations (Gonzalez et al., 2008; Roller, 2011;
Ziemann et al., 2011). In addition, no clear correlation of the probability of a positive in vitro test with
carcinogenicity was seen (Roller, 2011).
Recent studies with (nano)materials have shown, that cell proliferation in the lung as a consequence of
inflammation may be an important precursor of cancer. Histochemical analyses at Fraunhofer ITEM have
shown a high correlation between tumour frequencies and cell proliferation as well as genotoxicity in lung
epithelial cells (Rittinghausen et al., 2013) for carbon black, amorphous and crystalline silica. Another study
showed a relationship between tumour frequencies and inflammation, fibrosis, epithelial hyperplasia and
squamous metaplasia for amorphous and crystalline silica, carbon black and coal dust (Kolling et al., 2011).
These correlating parameters can be determined in studies of much shorter duration. The amount of these
studies with nanomaterials is considerably higher than the amount of long term studies and some of these
studies are conducted under non overload conditions with several dose levels. Therefore these studies
provide the possibility of deriving NOELs and LOELs of the nano-objects and the possibility of assessing
dose response and risks of nanomaterials as a basis for regulation.
In this research project repeated dose toxicity studies (including carcinogenicity studies) with different nanoobjects are analysed with the purpose to identify the precursors of carcinogenicity in these studies, to
compare the toxicity of different types of nano-objects and to compare the toxicity of nano-objects with
objects of larger size. Based on these analyses, already existing proposals for grouping of nanomaterials are
evaluated and extended.
For a better overview of the complex data studies were entered into the relational database PaFtox (Particle
and Fibre toxicity database). This approach allows comparison of many parameters including statistical
analyses.
1
1.2
Nanomaterials ---Definitions
Efforts have been made to develop a consistent terminology for nanomaterials. According to ISO/TR 12802:
2010 two types of nanomaterials are distinguished: nano-objects (external nanoscale dimension) and
nanostructured materials (internal nanoscale structure or surface structure). These are subdivided into further
subgroups. Nano-objects have at least one dimension below 100 nm and cover nanoparticles (3D nano),
nanofibres (2D nano) and nanoplates (1D nano) as well as nanocrystals (quantum dots, those exhibit sizedependent properties due to quantum confinement effects on the electronic states, see Fig 1). Nanofibres are
again subdivided into nanorods (solid fibre), nanotubes (hollow fibre) and nanowires (electrically conducting
or semiconducting nanofibres.). Nanotubes are currently distinguished between single-wall carbon nanotubes
(SWCNT) - consisting of a single cylindrical graphene layer, double-wall nanotubes (DWCNT) - composed
of two nested, concentric single-wall carbon nanotubes and multi-wall carbon nanotubes (MWCNT) composed of nested, concentric or near-concentric graphene sheets with interlayer distances similar to those
of graphite (ISO/TS 80004-3:2010).
Nanostructured materials have an internal or surface structure with a significant fraction of features, grains,
voids or precipitates in the nanoscale (like nanostructured powders, nanocomposites, nanodispersions,
nanoporous material). However, nanodispersions without any measureable interaction between nano-objects
and medium (the medium is just background) are actually considered as cluster/accumulation of nanoobjects. Articles that contain nano-objects or nanostructured materials are not necessarily nanostructured
materials themselves (ISO/TS 80004-4:2011).
Most of the commercially produced nanoproducts are aggregates and / or agglomerates of nano-objects,
which may be released under energy uptake (BAuA, 2007; BAuA/BfR/UBA, 2007; Creutzenberg et al.,
2012). Also, under typical experimental conditions, the majority of the nano-objects in the exposure milieu
are aggregated or agglomerated.
2
Fig 1:
Taxonomy of Nanomaterial; adapted from ISO/TR 12802:2010 & ISO/TS 80004-4:2011
The European Commission (EC, 2010) proposes to define a material as a nanomaterial when it has a specific
surface area by volume greater than 60 m2/cm3, excluding materials consisting of particles with a size lower
than 1 nm. The volume-specific surface area (VSSA) of a material is generally calculated from its bulk
density and its mass specific surface area. The latter is usually determined by gas absorption methodology
called the BET-method (Brunauer et al., 1938) that allows surface area or porosity measurements for
nanoparticles as small as 1 nm. From a 3D reconstruction of a nanomaterial, its surface area and its volume
can, in principle, be estimated directly, such that its VSSA can be calculated, even on a per particle basis
(Van Doren et al., 2011).
2
2.1
Methods
Literature search
2.1.1
Approach
The literature search was a tiered approach. First a most comprehensive literature search was performed. The
different terms used for nano-objects, possible relevant effects and the application routes (Tab. 1) were used
to perform a combined research in the databases Pubmed, Toxline and Web of Science.
3
Tab. 1:
Concept of literature search
Search for effects
Lung effects
Organ synonyms
Effects
Lung
toxicity
genotox*
airway
disorder
mutagen
pulmonary
disease
tumour
respirat*
function
fibrosis or fibrinogen
inflammation
cancer
carcinogenic or carcinoma
oxidative
mesotheliom*
damage
Search for nano-objects
nanoparticles
nanoparticle* / nano particle*
nanotube* / nanofibre* / nanowire*
nanoscale* particulates
nanomaterial*
fine particles
fine dust*
fine particle*
ultrafine particles
ultrafine dust*
ultrafine particle*
Search for application route
inhal*
(via air)
respirat*
airway
intratracheal
intraperitoneal
Search terms as “repeated or chronic or subchronic” were tested but led to very few hits as these words are
seldom used in the title, abstract or key words. The search results were collected in EndNote. Here, medical
related studies were excluded by searching for key words “drug delivery and “dry powder inhaler”. Based on
the abstracts, a preliminary list of potential relevant references was established. This list was subsequently
enhanced by cross references identified in the publications during the actual data entry in the database.
2.1.2
Results
The literature research led to 1343 hits in Pubmed, 1348 in Toxline and 1003 in Web of Science (January
2011). After pooling in EndNote, deleting the doubles and the medical related publications as well as
screening the abstracts, about 453 publications were ordered. A lot of these publications are reviews or minireviews which is consistent with current great interest in the scientific and regulatory community. However,
the reviews were also screened to find additional references. About one third are studies with study durations
from one to seven days which were separated for potential later projects. About 200 are potentially suitable
4
(see criteria under point 2.2.3) and up to now, 87 publications from 41 different institutions containing 131
studies were entered into the database.
After discussion with UBA, the following material types were selected for entry into the database
“Inert” particles (granular biopersistant dusts)
•
Carbon Black
•
Titanium dioxide
•
Aluminium oxide
Silicon dioxide
Heavy Metals (elemental or oxides)
•
Silver
•
Manganese
•
Nickel
•
Iron
•
Cerium
Carbon Nanotubes
•
Single wall carbon nano tubes
•
Multi wall carbon nano tubes
2.2 Data analysis by means of a relational database
2.2.1
Database Structure
The structure of the database is based on the already existing database for chemicals, RepDose
(www.fraunhofer-repdose.de, Bitsch et al., 2006) that has been developed at Fraunhofer ITEM. Particles and
fibres are characterised by other physico-chemical properties than chemicals. Therefore, the structure was
adapted and extended in this part of database (in the following Particle and Fibre toxicity, PaFtox database).
It was developed in Microsoft Access®. This software has been selected because it is commonly available
and can be easily handled also by non-experts. The database consists of three parts, the nano-object
characterisation, the study design and the effect related part. Generally, there are several possibilities for data
entry, picklist (fixed or expandable), free text or numerical figures.
For a comprehensive analysis of particle characteristics and toxic effects the database entries have to be
uniform and standardized. For this purpose glossaries were used for all fields, which can be addressed by
queries for all relevant information such as particle characterisation, study design and effect data.
Descriptions of identical observations differ between studies. As a general ontology for toxicological studies
is currently not available (Tcheremenskaia et al., 2012), the documentation of toxicological study data in a
database requires the development of a standardised vocabulary. For this purpose in the PaFTox database
observations, described by different terms were collected. If possible, one term was selected and entered into
the respective glossaries. Data entry guidance assures that the right synonyms are used subsequently for data
entry.
5
Glossaries were defined as picklists, e.g. for effects and targets. Picklists are further used to document:
surface property, sample preparation, distribution, unit of exposure/dose, species, strain, sex, application
route, parameter unit, score (severity of damage) and significance.
In contrast to the “defined fields” mentioned above “free text fields” are problematic, as users tend to include
typos and do not use standardised text, which hampers database queries. The current PaFTox database
therefore, does only include few “free text” fields. A typical example is the field “effect additional”, where
the user can enter details e.g. if more details on the location of the effect are available, one gender was more
sensitive than the other etc.
Fig 2:
Data entry mask of the PaFtox database
6
2.2.2
Particle and fibre characterisation
In the guidance on physico-chemical characterisation of engineered nanoscale materials for toxicological
assessment, it is stated that it is not sufficient to rely on a supplier’s commercial characterisation, as that
information is tailored to customer applications (ISO/TR 13014:2012). Nanodispersion (aerosol or liquid
dispersion) are usually instable as nano-objects (particles and fibres) tend to aggregate or agglomerate.
Therefore, it is recommended to test the material “as received” and “as administered“(ISO/TR 13014:2012).
Thus, there are three different types of data on nanomaterial characterisation, which all can be entered into
the database.
•
Specification of primary object by producer / supplier (as produced)
•
Specification of primary object by authors (as received)
•
Specification of secondary objects by authors (as administered, exposure media)
In line with ISO/TR 13014:2012, the following information can be entered into the database:
•
size / distribution
•
aggregation/agglomeration state in the exposure media
•
shape
•
specific surface area
•
composition / purity
•
surface chemistry
•
solubility / dispersibility
•
surface charge
2.2.3
Selection criteria for studies and materials
The particles and fibres which have been entered into the PaFtox database have been selected by availability
of suitable and reliable data. In general, five criteria were used for the selection of studies: application route,
nanoscale dimension, reliability, species and study duration.
The highest priority for entering into the database was given to inhalation studies (whole body or head/nose
only). As the number of inhalation studies is limited for nano-objects also intratracheal and pharyngeal
studies were included.
Generally studies with nanoscaled particles were preferred, but additionally larger particles and carbon
nanotubes were included for comparison reasons.
As guideline studies are currently seldom available, this criterion couldn’t be used for the selection. Nonguideline studies may address special questions. Therefore, the reliability of all publications entered in the
database was assessed based on internal quality criteria (similar to RepDose, (Bitsch et al., 2006)) and
resulted in reliability classes A (comprehensive study design and scope, high reliability) to C (quality not
accessible, preliminary).
The majority of repeated dose toxicity studies for nano-objects were performed in rats or mice. The study
selection was initially restricted to these two species, to get comparable data on target organs, mechanisms of
toxicity, and LOELs for different nano-objects.
7
In general, studies with study durations from 28 days up to lifetime exposure were selected. Occasionally,
shorter parallel studies of long-term studies (same publication) were entered into the database. For some
substances several studies were included in the database. This might be useful to cover different endpoints
and to analyse the influence of the study duration on several endpoints. Moreover, contradictory results can
be revealed by this selection strategy.
2.2.4
Study design
In the study part, information about the study design and exposure related information can be entered.
•
application type
•
exposure duration (hours per day, days, days per week)
•
instillation (number, frequency)
•
post-exposure duration
•
species/strain/sex
•
number of animals
•
particle and fibre characteristic as administered (secondary object)
•
reference
•
reliability
•
scope of study
This database contains four different application types, exposure via air (whole body and nose / head only)
and via instillations (intratracheal and pharyngeal). The airborne and instillation studies comprise a
fundamental different exposure regime. The standard exposure protocol for inhalation studies via air is 6
hours a day 5 days a week and 28, 91 or 730 days for subacute, subchronic or cancer studies. Instillation
studies are mostly performed with one instillation and the corresponding number of days for observation
(post-exposure in this database).
To enable a standardised data entry in the PaFtox database, one study is defined by the application type, the
exposure conditions and the species/strain. Due to this definition it occurs that one publication often contains
more than one study, but it happens also that one study was distributed over several publications. Therefore,
the number of publications in the database (appendix 7.1) is different from the number of studies.
Nano-objects in aerosols or in dispersions tend to aggregate or agglomerate. This tendency depends on
several physico-chemical properties of the nano-object and of the exposure media. A lot of information is
gathered in our report for the BAuA project F2133 (Schaudien et al., 2011). To enable retrospective
conclusions, not only the metric information (e.g. MMAD) of the administered objects is a data field of this
database but also the descriptive information on the exposure medium and conditions were collected.
As described under chapter 2.2.3, only data for rodents were entered into the database. Studies could have
been performed with both sexes and one sex. Two free text fields allow entering more study specific details:
the exposure additional and the study additional. The exposure additional describes more specific details of
the exposure regimen and the field study additional is related to the study design such as a special focus,
unexpected death in single dose groups, description of interim sacrifices (number of animals per time point).
8
The number of examinations performed in particle studies may differ considerably, because there are
currently no guidelines, which define the endpoints to be investigated. The comparison of toxicological
potency/effects in different studies is difficult, if it is not distinguished between absence of an effect and
absence of examination. Therefore, the scope of investigation is included in the database, where every single
investigation e.g. level of specific cytokine is documented.
2.2.5
Toxicological data
In any study a certain number of targets/organs is examined. In each target/organ numerous effects may be
investigated using different methods. The database aims to describe all toxicological effects observed in the
in vivo study. In PaFtox, effects are not related to the type of examination e.g. necropsy, histopathology or
organ weight, but assigned to the target/organ where it occurred. The standardized glossary of targets/organs
was adopted from the RepDose database. RepDose contains repeated dose studies of different exposure
duration with chemicals for oral and inhalation exposure. However in particle and fibre studies, lung is
mainly investigated but here more comprehensively as in similar chemical studies. Therefore, in addition to
the already documented targets of the respiratory tract in RepDose, PaFtox includes some more specific
targets, which are pleura and bronchio-alveolar lung fluid (BALF). The effect glossary was extended by new
effects, which are particle specific or are coming from specific examinations, e.g. cytokine levels that are not
common in repeated dose toxicity studies as they are not required in the corresponding guidelines. New
entries can easily be added to the glossary. Tab. 2 shows examples for effects covered in the glossary for
bronchio-alveolar lavage fluid (BALF) and lung.
It can be distinguished between general effects, which can occur in several organs, e.g. hyperplasia, collagen
or burden, and organ specific effects, e.g. bronchiolo-alveolar carcinoma (lung).
For each effect the lowest observed effect level (LOEL) is documented, as well as the sex being affected.
The field “effect additional” (2.2.1) further allows documenting some more details, if given in the study
report. Further the grading of the effect is documented in the newly developed effect level table.
This table may contain quantitative data (measured parameter), semiquantitative data (scores - mainly
histopathology) and qualitative data (yes/no). In any case, the information on dose, sex and time point is
given and the parameter level for quantitative data, the score levels minimal, mild, medium, severe, very
severe for semiquantitative data and ‘changed’ or ‘no change’ for qualitative data. If available, the
corresponding significances are entered. Sometimes effect data were only available in figures. In these cases
the values were estimated by a standardized read out system.
The detailed reports provided in the appendix 7.3 demonstrate the level of detail for each individual study.
2.2.6
Quality assurance
Every entry was double checked according to the 4 eyes principle by another scientist of the group. To
improve the speed of the data entry, an import module was programmed which allows combining of different
versions of the database. Further, for better overview it is possible to print a report on each study entered into
the database and on each material (see appendix 7.2 for examples).
Several queries have been made to assure consistency of entries e.g.:
•
Are all relevant fields filled with information? (E.g. all fields of study design)
•
Do doses correspond to effect LOEL values?
•
Do calculated values correspond in all fields of the database, e.g. number of decimals equal?
9
•
Are effects documented in a comprehensive way?
•
Are effects documented twice? If so, for which reasons?
•
Are effects in the glossary redundant?
•
Which effects are rare? Which grade of detail is needed?
•
Control for synonyms.
10
Tab. 2:
Examples for effects in BALF and lung
Target / Organ
Parameter / Effect
BALF
Total protein
Lactate dehydrogenase (LDH)
PMN
Total cells
Neutrophils
Alveolar macrophages
Lymphocytes
Leukocytes
Reactive oxygen species (RNS) ex vivo
Alkaline phosphatase (AP)
β-glucuronidase
Gamma-glutamyl transferase ( γ
-GT)
Burden
Tumour necrosis factor protein (TNF-α)
Reactive oxygen species (ROS) ex vivo
Lung
8-OHdG
Adenosquamous carcinoma
Alveolar bronchiolization
Clearance
Alveolar proteinosis
Apoptose
Benign cystic keratinizing squamous-cell tumour
Bronchiolo-alveolar adenocarcinoma
Bronchiolo-alveolar adenoma
Bronchiolo-alveolar carcinoma
Burden
Cell depletion
Cell division cycle mRNA (Cdc2a)
Cell proliferation
Changes in organ structure
Chemokine mRNA (CCL2)
11
2.2.7
Database queries
NormDose: Exposure conditions in inhalation studies differ in dose level, exposure duration in hours per day
and days per week. Therefore the original dose mostly provided for 6 h per day, 5 days per week in
inhalation studies was normalised to 24 h/day and 7 days/week.
CumDose: If instillation studies used more than one instillation, the time point of effect determination was
compared with the instillation regime and the corresponding cumulative dose for that time point was
calculated.
Categories for time points: The studies differ also regarding their time regime. Thus the determination of
effects occurred on a number of different days. Therefore, to simplify data analyses, the time points were
categorised.
Statistical analyses were performed with STATISTICA® and Microsoft excel®.
To better illustrate differences in numbers of studies or LOELs identified, the three colour code offered in
Excel was used and upper and lower limit (10th percentile, 90th percentile) were assigned to the traffic light
code; green means better results or more information and red means worse results or less information.
By analysing the minima of doses where an effect was positive, LOELs can be determined. Depending on
the conditions, chosen for the query, different LOELs have to be distinguished: effect-LOEL, organ-LOEL,
study-LOEL, substance-LOEL and object-LOEL or categories thereof. If several studies were available with
the same route and duration for a specific substance, for some analyses the lowest LOEL, i.e. the substanceLOEL was used.
12
3
Data analysis
3.1
Due to dimensional differences between nanoparticles and nanotubes the data about these two types of nanoobjects are analysed separately. Overall relatively little information about nano-objects characteristics is
provided in the toxicological studies entered in the PaFtox database. General no information is provided for
solubility, neither in water nor in the application media.
3.1.1
Specification of primary particles and fibres by producer / supplier (as produced) or by authors (as
received)
Overall, the amount of data related to primary characterisation is very low, see summary statistics about the
primary characterisation (Tab. 3).
Tab. 3:
Primary characterisation in studies with nano-objects
Specification by producer /
supplier
Specification by authors
Specification by
producer/supplier or author
Diameter (mean)
93
25
114
Diameter (min)
15
3
16
Diameter (max)
17
4
19
Inner diameter *
0
0
0
Outer diameter (mean)*
4
2
5
Outer diameter (min)*
3
3
6
Outer diameter (max)*
3
3
6
Length (mean)*#
5
2
5
Length (min) *#
4
2
5
Length (max) *#
3
1
4
Medium
2
8
9
Determination method
17
33
46
Distribution type
3
11
11
Cristal structure
63
16
79
Shape
15
16
29
Solubility
0
0
0
Specific surface
55
59
114
Surface property
27
23
44
Particle density
27
22
45
Overall number of studies: 131, *for tubes; # for tubes and rods
13
Tab. 4 demonstrates that even if information is available, there are differences for the frequently used
reference materials. Different batches or producing sites could cause these differences.
Tab. 4:
Available information for reference material ‘‘Min-U-Sil 5’’
Specification by supplier
Specification by authors
Diameter [nm]
Diameter [nm]
Median
Minimum
Maximum
Median
Minimum
Maximum
Kobayashi, et
al. 2009
Toxicology
Cites Warheit et al., 2007a and Kajiwara et
al., 2007
Kobayashi, et
al. 2010
Toxicology
Cites Warheit et al., 2006, 2007a,b;
Kobayashi et al., 2009
Shvedova , et
al. 2005
Am J Physiol Lung
Cell Mol Physiol
2140
Ogami, et al.
2009
Inhalation Toxicology
1600
10000
Warheit, et
al. 2004
Tox Sciences
1000
3000
Warheit, et
al. 2007a
Tox Sciences
300
2000
534
Warheit, et
al. 2007b
Toxicology
200
2000
480
14
300
700
3.1.2
Specification of secondary particles and fibres by authors (as administered)
Tab. 5 shows the number of studies where information on secondary characterization (as administered) was
available. No information was provided in the publications about solubility in the used medium, the
isoelectric point, the conductivity and almost nothing about zeta potential or spectra data which allow some
inferences to the dimension and appearance of the nano-objects in the media.
Tab. 5:
Secondary characterisation in studies with nano-objects
Number of studies
Inhalation studies:
Number of available
data
Percentage of
Inhalation studies
(n=38)
Instillation studies:
Number of available
data
Percentage of
Instillation studies
(n=93)
Diameter (median)
26
68%
24
26%
Diameter (GSD)
23
61%
1
1%
Diameter (min)
3
8%
9
10%
Diameter (max)
3
8%
10
11%
Medium
4
11%
31
33%
Determination method
26
68%
31
33%
Sample treatment
2
5%
66
71%
Application medium
2
5%
90
97%
Dispersant
0
0%
26
28%
Bulk density
4
11%
0
0%
Distribution type
38
100%
92
99%
Zetapotential
2
5%
1
1%
Solubility in medium
0
0%
0
0%
Isoelectric point
0
0%
0
0%
Conductivity
0
0%
0
0%
Diameter means mass median aerodynamic diameter and hydrodynamic diameter for inhalation and instillation studies, respectively.
A comparative analysis between MMAD or hydrodynamic diameter and primary diameter was performed
(Fig 3). A dependency could not be identified. However, MMADs cover several orders of magnitudes.
MMAD is a double critical parameter, first its size determines the deposition behaviour and fate (Oberdörster
et al., 2005) and second it’s difficult to standardize these size measurements, currently three different
techniques lead to three different results (Pauluhn, 2010). This issue of primary nano diameter versus micro
MMAD or hydrodynamic diameter contains the problem that in many studies the nano-objects are applied as
larger aggregates, only slightly different from original larger particles. As disaggregation seldom occurs,
only minor differences in effects could be expected between nano and larger particles.
15
Fig 3:
MMAD in inhalation studies (left) and hydrodynamic diameter in instillation studies (right)
3.2 Studies included
3.2.1
Routes and study durations
In the public literature a huge variety of study designs are published. Tab. 6 and Tab. 7 give an overview of
the design of inhalation and instillation studies. The inhalation studies differ with respect to the type of
inhalation (nose / head only), study duration, duration of postexposure period and duration of exposure per
day and per week. Similarly instillation studies differ with respect to frequency of instillations and time after
instillation. The study designs encountered most frequently are highlighted in bold. Within the different
studies, investigations have been performed at interim time points, especially in instillation studies.
16
Tab. 6:
Study design of inhalation studies
route
Category of
study duration
Exposure
duration in
days
Post-exposure
in days
Exposure time
in h/d
Exposure
duration in
d/wk
Number of
studies
Nose / head
only
Subacute
23
90
6
5
2
28
91
6
5
1
28
546
0.66
7
1
90
1
6
5
1
91
180
6
5
1
10
0
6
7
2
24
0
6
5
1
28
0
2
3
2
28
0
6
5
1
28
2
6
5
1
84
364
6
5
2
91
0
6
5
1
91
224
6
5
1
91
240
6
5
1
91
335
6
5
3
91
364
6
5
8
152
1
5
5
1
411
288
18
5
2
547
180
18
5
1
547
183
18
5
1
730
0
6
5
1
730
50
16
5
1
730
180
18
5
1
730
182
18
5
1
Subchronic
Whole body
Subacute
Subchronic
Chronic
Cancer
17
Tab. 7:
Study design of instillation studies
Application
route
Category of
study
duration
Exposure
duration in
days
Postexposure in
days
Number of
instillation
Intratracheal
Cancer
1
874
1
28
872
5
1
wk
5
63
812
10
1
wk
1
63
827
10
1
wk
1
63
837
10
1
wk
4
64
826
10
1
wk
1
98
802
15
1
wk
1
105
695
16
1
wk
2
133
767
20
1
wk
2
203
697
30
1
wk
1
406
469
30
2
wk
1
1
98
1
2
1
181
1
6
1
274
1
1
2
456
2
1
d
3
63
393
10
1
wk
1
84
77
4
0.5
d
1
266
14
20
2
wk
1
1
3
1
1
1
7
1
1
1
13
1
1
7
21
2
1
wk
1
8
2
2
1
wk
1
21
0
15
5
wk
1
21
7
4
1
wk
1
1
28
1
8
1
30
1
2
1
42
1
4
1
60
1
6
1
90
1
17
35
0
6
1
wk
2
35
1
6
1
wk
2
42
0
30
5
wk
1
42
1
7
1
wk
1
Chronic
Subacute
Subchronic
18
frequency
per
Number of
studies
1
Application
route
Pharyngeal
3.2.2
Category of
study
duration
Subchronic
Exposure
duration in
days
Postexposure in
days
Number of
instillation
frequency
per
Number of
studies
61
30
3
1
mo
3
63
0
45
5
wk
1
1
28
1
1
1
60
1
3
Species
Usually studies were performed with rats or mice (Tab. 8). Due to the high spontaneous lung tumour rate in
mice, the rat is the preferred species especially for long term studies with particles. This is also reflected by
the species distribution in the database where 80% are rat studies. However, especially for the shorter study
duration, where inflammation endpoints are mainly investigated, mice are also used (see Tab. 8).
Tab. 8: Types of studies in rats and mice
Category of study duration
Application route
Subacute
Nose /head only
Subchronic
Number of mouse studies
4
Whole body
3
4
Intratracheal
7
12
Pharyngeal
1
Nose /head only
Chronic
Total number species
Number of rat studies
2
Whole body
3
13
Intratracheal
5
32
Pharyngeal
3
Whole body
3
6
Intratracheal
1
32
All routes
26
105
Bold: Highest number of studies
3.2.3
Dose levels, NOELs and LOELs
According to the respective OECD guidelines repeated dose toxicity studies are performed with 3 dose levels
and a control group. The lowest dose group is intended to show no effects (NOEL), the medium dose group
should reveal slight toxicity (LOEL) and the highest dose group should have clear toxic effects, but no
increased mortality. For toxicological evaluations effects at the LOEL are most important, as these reflect
effects at low dose levels. As demonstrated in Tab. 9, 74 studies investigated only one dose level, 22 studies
two dose levels and 35 studies used three or more dose levels.
19
Tab. 9:
Number of different dose levels
Number of studies
Route
Category time
point
1 Dose Level
2 Dose Levels
3 and more dose
levels
Nose /head only
37-99
1
Nose /head only
100-189
3
Nose /head only
190-365
1
Nose /head only
366-912
1
Whole body
1-10
2
Whole body
11-36
4
Whole body
37-99
Whole body
100-189
1
Whole body
190-365
1
Whole body
366-912
12
Intratracheal
1-10
1
Intratracheal
11-36
10
4
4
Intratracheal
37-99
14
12
9
Intratracheal
100-189
5
1
1
Intratracheal
190-365
2
Intratracheal
366-912
19
4
1
Pharyngeal
11-36
Pharyngeal
37-99
1
1
1
1
8
2
1
2
1
3.3 Particles included
Currently, the PaFtox database contains 17 different materials. For titan dioxide and Carbon Black numerous
studies with different particle sizes were available. An overview is provided in Tab. 10.
Tab. 10:
Particles included
Substance
Titan dioxide
Carbon Black
Diameter
(mean) in nm
4.9
2025
154
180
200
250
1000
14
15
37
56
70
95
120
260
Specific
Surface in
m2/g
316
3266
10
9.9
8.8
6.5
2.34
271337
230
43
45
37
22
n.g.
n.g.
Not all particles are available for all application routes
The distribution of particles over the different application routes is illustrated in Tab. 11.
20
Tab. 11:
Number of studies for different substances and particle diameter categories and application routes
Number of studies
Substance
Category of diameter
(mean)in nm
Intratracheal
Nose/ head
only
Aluminium oxide C
50
2
Aluminium trioxide
50
1
Aluminium trioxide
500
1
Aluminium oxyhydroxide
50
Carbon Black
50
13
Carbon Black
100
1
1
Carbon Black
500
1
1
Cerium(IV) oxide
50
1
Iron(II,III) oxide
10
1
lamp black 101+diesel soot
100
3
Manganese(IV) oxide
50
3
Nickel
50
1
Nickel hydroxide
50
Nickel oxide
50
2
Nickel oxide
3000
1
Silicon dioxide
50
16
Silicon dioxide
500
6
Silicon dioxide
1500
8
Silicon dioxide
3000
1
Silicon dioxide
5000
1
Silicon dioxide
8000
1
Silver
50
2
Titanium dioxide
10
4
Titanium dioxide
50
11
7
Titanium dioxide
500
6
5
Titanium dioxide
1500
1
Titanium dioxide
3000
Toner
500
Pharyngeal
Whole body
1
7
2
1
2
4
1
1
1
Total number of studies: 122
3.4 Parameter / Effect analysis
3.4.1
Target / organs and effects
Fig 4 presents an overview of affected targets / organs, distinguished between the different application
routes. Generally, lung and BALF are the main targets, followed by lymph nodes.
21
Fig 4:
Overview effected targets / organs
Tab. 12 provides an overview of the effects found in all studies, irrespective of the route of application, dose
and time point. It shows that some effects appear very frequently, such as lactate dehydrogenase (LDH), total
protein, PMN in BALF or macrophage infiltration, fibrosis and inflammation in the lung.
22
Tab. 12:
Observed effects
Target / Organ
Parameter / Effect
Parameter / Effect observed (no of studies)
BALF
50
BALF
Total protein
50
BALF
PMN
50
Lung
Macrophage infiltration
48
Lung
Weight
46
BALF
Total cells
41
BALF
Neutrophils
40
BALF
36
Lung
Fibrosis
34
Lung
Inflammation
28
Lymph node
Burden
28
BALF
Lymphocytes
24
Lung
Hyperplasia
22
Body weight
Weight decreased
21
Lung
Collagen
20
Lung
Bronchiolo-alveolar adenoma
18
Lung
Tumour (other)
17
Lung
Squamous cell carcinoma
16
Lung
Alveolar type II cells
16
Lung
Granuloma
16
Lung
15
Lung
Cell proliferation
15
BALF
Leukocytes
13
Lung
Macrophages foamy
13
Lung
Bronchiolo-alveolar carcinoma
13
Lung
Cystic keratinizing epithelioma
13
Total number of studies: 131
As indicated in Fig 4 most effects are local effects in the respiratory tract. Tab. 13 shows that effects in the
lymph nodes, spleen, blood and pleura have been found especially in inhalation studies and indicate
migration of the nano-objects.
23
Tab. 13:
Parameter / Effects beyond the respiratory tract
Target / Organ
Effect
Nose / head only
Whole body
Intratracheal
Adrenal gland
Weight
Brain
Burden
Brain
Functional disorders
1
Clinical symptoms
Behaviour abnormal
1
Blood
B cells
3
Blood
Erythrocytes
Blood
Granulocytes
Blood
Haematocrit
2
Blood
Haemoglobin
2
Blood
Leukocytes total
1
Blood
Lymphocytes total
3
Blood
Natural killer cells (NK)
3
Blood
Natural killer T cells (NKT)
3
Blood
Neutrophils total
Blood
T cells
3
Blood
T cells CD4+/CD8+
3
Heart
Blood pressure
2
Heart
Heart rate
1
Kidney
Burden
1
Liver
Burden
2
Liver
Hyperplasia
1
Liver
Necrosis
2
Liver
Vacuolization
1
Liver
Weight
Lymph node
Burden
Lymph node
Cell proliferation
Lymph node
4
Lymph node
Discoloration
2
Lymph node
Fibrosis
Lymph node
Granuloma
1
5
Lymph node
Histiocytosis
3
1
Lymph node
Hypercellularity
1
Lymph node
Hyperplasia
2
Lymph node
Hypertrophy
3
Lymph node
Infiltration
8
1
2
1
8
1
2
6
2
25
40
23
1
1
8
24
4
6
Target / Organ
Effect
Lymph node
Inflammation
Lymph node
Macrophage accumulation
Lymph node
Macrophage damage
Lymph node
Lymph node
Weight
Nervous system
Pleura
Pleura
Collagen
6
Pleura
Fibrosis
1
Pleura
Inflammation
Pleura
Thickening
Spleen
Burden
Spleen
Weight
Thymus
Weight
1
Urine
Protein
1
Vascular system
Burden
1
3.4.2
Nose / head only
Whole body
Intratracheal
4
9
3
7
3
4
13
1
1
1
1
1
4
1
1
Reversibility of effects
Tab. 14 documents the effects per target organ for studies with whole body exposure which include in
addition to the investigations at the end of the exposure period also investigations after a postexposure
period. The postexposure durations ranged from 2 to 364 days.
In total 755 effects were observed, 407 effects within the treatment period of the studies and 348 effects in
addition in the post exposure period. The number of effects (at lowest observed effect level) were counted
per time point and are subdivided into two groups: effect observed within exposure duration (termed (-)
postexposure) or the effect was observed in the postexposure time (termed (+) postexposure). In most studies
interim sacrifices were performed, so that effects for several time points can be distinguished. Time points
were grouped into six time categories, evaluations <= 10 days (termed 10 days), <=36 days (termed 28 days),
<100 (termed 90) <= 189 days (termed 182 days), <= 365 days (termed 365 days) and > 365 days (termed
700 days). All effects of the treatment period were also observed in the postexposure period. To obtain
comparable results for the different groups, the number of positive effects was normalized to the number of
studies of the corresponding subgroup.
25
Tab. 14:
Parameter / Effect of study evaluation (+) or (-) postexposure in whole body inhalation studies
Percentage of positive effects per total study number
(n = 22)
(-) postexposure
Target / Organ
Parameter / Effect
90
182
BALF
AM relative
23
BALF
AM total
9
BALF
Burden
9
BALF
LDH
23
BALF
Neutrophils relative
14
BALF
Neutrophils total
9
BALF
PMN relative
27
BALF
PMN total
9
BALF
ß-glucuronidase
23
BALF
Total cells
41
BALF
Total protein
23
Body weight
Weight
5
Clinical symptoms
Mortality
Lung
14
Lung
mRNA (CCL2)
9
Lung
Deposits
Lung
Fibrosis
Lung
Hyperplasia alveolar
type II cells
23
Lung
Infiltration
23
Lung
Inflammation
9
Lung
Macrophage damage 14
Lung
Macrophage
infiltration
32
9
Lung
Macrophages
interstitial
9
5
Lung
Metaplasia
Lung
Weight
27
Lymph node
Burden
Nose
Ratio (+)postexposure /
(-)postexposure
(+) postexposure
365
5
700
23
90
182
365
700
90
23
32
9
23
1
9
9
9
45
1
5
5
1
9
5
18
5
5
9
23
14
9
32
1
14
18
9
14
1
9
18
9
5
1
27
14
5
14
1
5
18
1
9
9
18
23
9
9
32
1
41
9
5
14
1
9
23
9
5
23
1
23
5
9
18
45
1
27
5
9
182
365
700
2
2
2
1.75
2
2
2
2.5
2
41
14
9
5
27
9
5
5
5
5
14
1.75
2
1.5
1
1
3
1
1.5
1
5
9
14
18
27
5
32
32
68
1
2.33
1.75
2.5
5
5
14
23
14
5
32
1
3
1
2.3
5
5
23
14
9
18
1
2
4
5
14
9
5
5
18
1
1
1.3
9
14
9
23
1
9
23
32
50
27
50
1
5.5
3
2.2
9
9
9
27
23
32
1
6
2.5
3.5
5
9
5
14
1
1.5
14
23
32
27
23
23
45
1
1.67
1
1.4
27
5
5
27
27
18
23
45
1
4
5
1. 7
Degeneration
5
18
18
18
5
18
18
45
1
1
1
2.5
Nose
Hyperplasia
5
9
9
5
14
9
18
1
1.5
1
Nose
Inflammation
9
18
9
27
1
1.5
Nose
Squamous cell
metaplasia
9
18
9
27
1
1.5
5
5
26
1
2.5
Traffic light code used for illustration lower limit: 10 percentile set to green and upper limit: 90 percentile set to red;
AM - alveolar macrophages
27
Tab. 14 also depicts in the last column the ratio between the number of effects during the treatment and
postexposure period of studies. If this value is higher than 100% it means, that effects are occurring more
frequently at the LOEL of the study in the subgroup with postexposure.
No effect disappeared, indicating that no effect was fully reversible. This analysis does however, not specify
differences in grade and severity of effects and thus their in- or decrease during the postexposure period. In
addition one effect in BALF and eight effects in lung are only seen in the postexposure period (Tab. 15).
Tab. 15:
Parameter / Effects (number) appearing only in the postexposure period at LOEL per time point in studies with
whole body exposure.
Category time point
Target / Organ
Parameter / Effect
100-189
366-730
BALF
Glutathione peroxidase
1
2
Lung
Adenosquamous carcinoma
1
Lung
Benign cystic keratinizing squamous-cell tumour
2
Lung
Bronchiolo-alveolar adenocarcinoma
3
Lung
Gamma-glutamylcysteine synthetase (γ-glutamGCL)
2
Lung
Manganese superoxide dismutase (Mn SOD)
2
Lung
Squamous metaplasia
1
Lung
Thickening
1
Lung
Tumour supressor protein p53
1
3.4.3
Parameters affecting toxicity of particles and fibres
In a preliminary analysis the influence of different parameters discussed to influence the toxicity of nanoobjects was investigated. The following parameters were analysed:
•
the composition of the (nano) particle
•
the mean diameter
•
the specific surface
Studies with whole body exposure and intratracheal studies were analysed, because most studies were
available for these application types. In most studies interim sacrifices were performed, so that effects for
several time points can be distinguished. Time points were again grouped into six time categories,
evaluations <= 10 days (termed 10 days), <=36 days (termed 28 days), <100 (termed 90) <= 189 days
(termed 182 days), <= 365 days (termed 365 days) and > 365 days (termed 700 days).
While in intratracheal studies investigations are already performed at early time points, the study design of
inhalation studies focus on analyses at later time points. However in both routes, not every time point is
covered.
28
3.4.3.1
Composition
Tab. 16 shows LOELs derived from different studies for different substances and for the different
application routes. There are considerable differences in toxicity between substances, while the exposure
duration has a minor influence on the toxicity.
Tab. 16:
LOELs per route, substance and time point
Application route
Nose / head only
Name
Aluminum oxyhydroxide
Min LOEL per time point
10
28
90
182
5E+00
4E+00
5E+00
6E-01
2E-02
2E-02
8E-02
MWCNT
Whole body
7E-01
9E+00
9E+00
3E+01
Carbon Black
3E+00
2E-01
2E-01
1E+00
2E-01
MWCNT
6E+00
Nickel hydroxide
2E-02
2E-02
Silicon dioxide
2E-01
2E-01
2E-01
2E-01
9E-02
9E-02
9E-02
Titanium dioxide
6E+00
9E-05
9E-03
4E-01
9E-02
Aluminium oxide C
1E+02
Aluminium trioxide
2E+00
2E+00
2E+00
Carbon Black
2E-01
1E+01
4E-01
Cerium(IV) oxide
2E-01
5E-01
Iron(II,III) oxide
3E-01
1E+00
7E+01
1E+02
2E+02
1E+01
1E+02
Manganese(IV) oxide
Pharyngeal
700
Silicon dioxide
Silver
Intratracheal
365
6E+01
1E+02
4E-02
2E-01
MWCNT
4E-02
4E-02
Nickel
5E-01
5E+00
Nickel oxide
3E-01
3E-01
3E-01
3E-01
Silicon dioxide
8E-03
8E-01
8E-01
5E+00
SWCNT
1E+00
5E+00
1E+00
Titanium dioxide
8E-01
8E-01
8E-01
Toner
2E+02
2E+02
2E+02
SWCNT
3E-01
5E-01
5E-01
1E+01
1E+01
1E+01
LOELs normalised doses for inhalation studies in mg/m3 and cumulative doses in mg/kg bw for instillation studies; Traffic light code used for
illustration lower limit: 10 percentile set to red and upper limit: 90 percentile set to green
To further investigate the influence of particle composition; effects were identified that were either caused by
reference particles (titanium oxide, silicon oxide, aluminium oxides and Carbon Black) or by heavy metals
and oxides (silver, nickel and oxide, iron oxide, cerium oxide and manganese oxide). Reference particles
cause overall 178 different effects (data not shown). The high number of effects is probably due to the fact
29
that especially Carbon Black and titanium dioxide are well investigated with numerous endpoints. A lower
number of unique effects have been observed for the heavy metal based particles (Tab. 17). Interestingly,
only one effect is caused by two different substances, but both are Nickel compounds. All other effects are
unique per substance.
Tab. 17:
Parameter / Effects occurring only with heavy metals and oxides
Target / Organ
Parameter / Effect
Substance
Adrenal gland
Weight
Manganese(IV) oxide
BALF
Apoptosis
Cerium(IV) oxide
BALF
Arginase-1 mRNA ex vivo
Cerium(IV) oxide
BALF
Caspase protein (CASP3) ex vivo
Cerium(IV) oxide
BALF
Caspase protein (CASP9) ex vivo
Cerium(IV) oxide
BALF
Cell cycle: G1 phase
Iron(II,III) oxide
BALF
Cell cycle: S phase
Iron(II,III) oxide
BALF
Chemokine mRNA (CCL3)
Nickel oxide
BALF
Chemokine protein (CCL2)
Nickel hydroxide
BALF
Nickel oxide
BALF
Cytokine-induced neutrophil chemoattractant protein (CINC-1)
Nickel oxide
BALF
Cytokine-induced neutrophil chemoattractant protein (CINC-2
BALF
Interleukin protein (IL-1)
Iron(II,III) oxide
BALF
Iron(II,III) oxide
BALF
Interleukin protein (IL-12) ex vivo
Cerium(IV) oxide
BALF
Iron(II,III) oxide
BALF
Iron(II,III) oxide
BALF
Lipid peroxides (LPO)
Nickel
BALF
Nuclear factor mRNA (NF-κB) ex vivo
Cerium(IV) oxide
BALF
Phospholipids
Cerium(IV) oxide
BALF
Suppressor of chemokine signalling mRNA (SOCS-1) ex vivo
Cerium(IV) oxide
Brain
Manganese(IV) oxide
Clinical chemistry
Calcium
Silver
Clinical chemistry
IgE
Iron(II,III) oxide
Clinical chemistry
Iron(II,III) oxide
Clinical chemistry
Iron(II,III) oxide
Clinical chemistry
Iron(II,III) oxide
Clinical chemistry
Iron(II,III) oxide
Clinical chemistry
Total protein
Silver
Clinical chemistry
Tumour necrosis factor protein (TNF-α)
Iron(II,III) oxide
Clinical symptoms
Behaviour abnormal
Manganese(IV) oxide
30
αβ)
Nickel oxide
Target / Organ
Parameter / Effect
Substance
Blood
B cells
Iron(II,III) oxide
Blood
Natural killer cells (NK)
Iron(II,III) oxide
Blood
Natural killer T cells (NKT)
Iron(II,III) oxide
Blood
T cells
Iron(II,III) oxide
Blood
T cells CD4+/CD8+
Iron(II,III) oxide
Liver
Hyperplasia
Silver
Liver
Necrosis
Silver
Liver
Vacuolization
Silver
Liver
Weight
Manganese(IV) oxide
Lung
Cytokine-induced neutrophil chemoattractant protein (CINC-1)
Nickel oxide
Lung
Cytokine-induced neutrophil chemoattractant protein (CINC-2
Lung
Glycoprotein mRNA (OPN)
Cerium(IV) oxide
Lung
Heat shock protein mRNA (HSP1a)
Iron(II,III) oxide
Lung
Heat shock protein mRNA (HSP8)
Iron(II,III) oxide
Lung
Interleukin mRNA (IL-1 α)
Nickel hydroxide
Lung
Interleukin protein (IL-1 α)
Nickel oxide
Lung
Nickel oxide
Lung
Lipidosis
Cerium(IV) oxide
Lung
Matrix metalloproteinase mRNA (MMP-12)
Iron(II,III) oxide
Lung
Iron(II,III) oxide
Lung
Iron(II,III) oxide
Lung
MHC II mRNA (H2-Eb1)
Iron(II,III) oxide
Lung
MHC II mRNA (H2-T17)
Iron(II,III) oxide
Lung
MHC II mRNA (H2-T23)
Iron(II,III) oxide
Lung
Minute volume
Silver
Lung
Peak inspiration flow
Silver
Lung
PMN total
Cerium(IV) oxide
Lung
Proteinase inhibitor mRNA (SLPI)
Iron(II,III) oxide
Lung
Serum amyloid mRNA (SAA3)
Iron(II,III) oxide
Lung
Tidal volume
Silver
Nervous system
Manganese(IV) oxide
Urine analysis
Protein
Silver
αβ)
Nickel oxide
Silicon dioxide is frequently used as reference material in toxicity studies of particles. Therefore we analysed
if there are silicon dioxide specific effects. Tab. 18 shows silicon dioxide specific effects and the size and
31
crystal structure responsible for the effect. Both types can cause significant effects in the lung, pleura and
lymph nodes of different severity. The weight gain of thymus found for crystalline silica was transient.
32
Tab. 18:
Parameter / Effects occurring only with silicon dioxides
Target / Organ
Parameter / Effect
Size & Crystal structure
Haematology
Neutrophils total
Nano amorphous or micro crystalline
Lung
Spongiosis
Lung
Swelling
Lymph node
Fibrosis
Lymph node
Infiltration
Lymph node
Inflammation
Nose
Necrosis
BALF
Chemotaxis
Micro crystalline
BALF
Phosphatidylglycerol/phosphatidylinositol
Micro crystalline
(PG/PI)
Bronchi
Hyperplasia mucus cell
Micro crystalline
Clinical chemistry
Alanine aminotransferase (ALAT)
Micro crystalline
Clinical chemistry
Alkaline phosphatase
Micro crystalline
Lung
8-oxoGua
Micro crystalline
Lung
Hyperplasia alveolar type II cells
Micro crystalline
Lung
p53 mutations
Micro crystalline
Lymph node
Hypertrophy
Micro crystalline
Pleura
Fibrosis
Micro crystalline
Thymus
Weight
Micro crystalline
Bronchi
Apoptosis
Nano amorphous
Bronchi
Necrosis
Nano amorphous
Clinical chemistry
Globulin
Nano amorphous
Clinical chemistry
Histamine
Nano amorphous
Clinical symptoms
Respiratory distress
Nano amorphous
Haematology
Haematocrit
Nano amorphous
Haematology
Haemoglobin
Nano amorphous
Lung
Chemokine protein (CXCL2)
Nano amorphous
Lung
Interferon gamma mRNA (IFN-γ)
Nano amorphous
Lung
Interleukin mRNA (IL-4, IL-8, IL-10, IL-13)
Nano amorphous
Lung
Interleukin protein (IL-1β, IL-4, IL-6, IL-8, IL-
Nano amorphous
33
Target / Organ
Parameter / Effect
Size & Crystal structure
10, IL-13)
Lung
Laminin
Nano amorphous
Lung
Matrix metalloproteinase mRNA (MMP-9,
Nano amorphous
MMP-10)
Lung
Matrix metalloproteinase protein (MMP-9)
Nano amorphous
Lung
Metallopeptidase inhibitor protein (TIMP-1)
Nano amorphous
Lung
Necrosis
Nano amorphous
Lymph node
Cell proliferation
Nano amorphous
Lymph node
Histiocytosis
Nano amorphous
Lymph node
Macrophage damage
Nano amorphous
Lymph node
Nano amorphous
Pleura
Nano amorphous
Crystalline covers also mainly crystalline.
3.4.3.2
Tubes versus particles
To investigate also the influence of the shape on toxicity an analysis was performed if carbon nano tubes
(CNTs) cause other effects than titanium oxide, silicon oxide, aluminium oxides, Carbon Black or heavy
metal compounds. Tab. 19 shows effects identified only for tubes but not with „inert“ particles, silicon
dioxide or heavy metal based particles. Interestingly, all tube specific effects are unique per CNT type.
Besides changes in cytokine levels other parts of the respiratory tract such as nose, pharynx and trachea are
affected. Further thickening of the pleura was found, an effect that is consistent with the hypothesis that
carbon nanotubes migrate to the pleura like asbestos fibres.
Tab. 19:
Parameter / Effects occurring only with carbon nanotubes
Target / Organ
Parameter / Effect
Substance
BALF
Collagen
MWCNT
BALF
Growth factor protein (TGF-β1)
SWCNT
BALF
SWCNT
BALF
Myeloperoxidase (MPO)
SWCNT
BALF
Reactive oxygen species (ROS)
SWCNT
Bronchi
Hypertrophy
MWCNT
Bronchi
Weight
SWCNT
Blood
Granulocytes
MWCNT
Blood
Leukocytes total
MWCNT
Larynx
MWCNT
Lung
SWCNT
34
Target / Organ
Parameter / Effect
Substance
Lung
SWCNT
Lung
SWCNT
Lung
Cytokeratin
SWCNT
Lung
Expiratory time
SWCNT
Lung
Glutathione (GSH)
SWCNT
Lung
Histiocytosis
MWCNT
Lung
Interferon gamma protein (IFN-γ)
SWCNT
Lung
Interleukin protein (IL-17A)
SWCNT
Lung
SWCNT
Lung
SWCNT
Lung
Pentosidine
SWCNT
Lymph node
Hypercellularity
MWCNT
Nose
MWCNT
Nose
Eosinophilic structures
MWCNT
Nose
Infiltration
MWCNT
Pharynx
Hypercellularity
MWCNT
Pharynx
Mucus
MWCNT
Pleura
Thickening
MWCNT
Trachea
Hypercellularity
MWCNT
Trachea
Mucus
MWCNT
3.4.3.3
Diameter and specific Surface
There is concern, that particles with nanoscale diameter have a higher toxicity than particles of larger scale,
due to their higher surface, inter alia. Therefore, we investigated, whether we can reproduce these results
with queries in the PaFtox database.
The LOELs were analysed for up to four different subgroups regarding their diameter and for two different
subgroups regarding their specific surface for six categories of study durations from 10 to 700 days in whole
body and intratracheal studies. The results are presented in Tab. 20 for the different diameters and in Tab. 21
for different specific surfaces. Generally the LOELs are lower for the particles with the smaller diameter and
the higher specific surface. The difference in LOEL for the study with the lowest LOEL for the respective
substances and time point is up to two orders of magnitude. The LOELs differ for particles within the same
diameter or surface category, confirming substance specific toxicity. Silver nanoparticles are by far the most
toxic nanoparticles.
35
Tab. 20:
Influence of particle diameter on LOELs
Application route
Nose / head only
Whole body
Substance
Diameter
10
28
90
182
5E+00
4E+00
5E+00
6E-01
2E-02
2E-02
8E-02
< 56 nm
MWCNT
< 56 nm
Silicon dioxide
< 56 nm
7E-01
9E+00
9E+00
3E+01
Carbon Black
< 56 nm
9E+00
2E-01
2E-01
1E+00
> 56 nm
3E+00
9E+00
9E+00
MWCNT
< 56 nm
6E+00
Nickel hydroxide
< 56 nm
2E-02
2E-02
Silicon dioxide
< 56 nm
2E-01
2E-01
2E-01
2E-01
> 56 nm
1E+01
1E+01
1E+01
1E+01
9E-02
9E-02
9E-02
9E-02
2E+00
2E+00
2E+00
2E+00
Silver
< 56 nm
Titanium dioxide
< 56 nm
9E-05
6E+00
4E-01
< 56 nm
Aluminium trioxide
< 56 nm
2E+00
2E+00
2E+00
> 56 nm
2E+00
2E+00
2E+00
< 56 nm
2E-01
1E+01
4E-01
7E+01
> 56 nm
6E+00
2E+01
7E+01
Cerium(IV) oxide
< 56 nm
2E-01
5E-01
Iron(II,III) oxide
< 56 nm
3E-01
1E+00
Lamp black 101+diesel soot
> 56 nm
Manganese(IV) oxide
< 56 nm
MWCNT
< 56 nm
Nickel
Nickel oxide
9E+00
1E+02
1E+02
2E+02
1E+01
1E+02
6E+01
1E+02
4E-02
4E-02
4E-02
2E-01
< 56 nm
5E-01
5E+00
< 56 nm
3E-01
3E-01
3E-01
3E-01
7E+00
7E+00
7E+00
> 56 nm
Silicon dioxide
2E-01
9E-03
Aluminium oxide C
Carbon Black
Pharyngeal
700
Aluminium oxyhydroxide
> 56 nm
Intratracheal
365
< 56 nm
8E-03
8E-01
1E+00
> 56 nm
1E+00
8E-01
8E-01
SWCNT
< 56 nm
1E+00
5E+00
1E+00
Titanium dioxide
< 56 nm
8E-01
8E-01
8E-01
4E+01
> 56 nm
5E+00
5E+00
2E+01
1E+01
Toner
> 56 nm
2E+02
2E+02
2E+02
SWCNT
< 56 nm
3E-01
5E-01
5E-01
5E+00
5E+01
7E+01
1E+01
1E+01
36
Tab. 21:
Influence of specific surface on LOELs
Application route
Nose / head only
Name
Category
specific
surface
400
MWCNT
400
Silicon dioxide
70
10
28
90
182
5E+00
4E+00
5E+00
6E-01
2E-02
2E-02
8E-02
700
1E+00
1E+00
1E+00
1E+00
2E-01
2E-01
1E+00
2E-01
7E-01
70
Whole body
365
Carbon Black
400
9E+00
MWCNT
400
6E+00
Nickel hydroxide
70
2E-02
2E-02
70
1E+01
1E+01
1E+01
1E+01
Silicon dioxide
400
2E-01
2E-01
2E-01
2E-01
Titanium dioxide
70
9E-02
9E-02
9E-02
9E-02
Aluminium oxide C
400
Aluminium trioxide
400
6E+00
4E-01
1E+02
2E+00
70
2E+00
400
2E-01
1E+01
Cerium(IV) oxide
70
2E-01
5E-01
70
Manganese(IV) oxide
70
MWCNT
70
4E-02
4E-02
Nickel
70
5E-01
5E+00
70
7E+00
400
Nickel oxide
Silicon dioxide
2E+00
1E+01
Carbon Black
1E+02
7E+01
4E-01
7E+01
2E+02
1E+01
1E+02
4E-02
2E-01
7E+00
7E+00
7E+00
3E-01
3E-01
3E-01
3E-01
70
1E+00
8E-01
8E-01
5E+00
400
8E-03
8E-01
1E+00
70
8E-01
8E-01
8E-01
Intratracheal
Titanium dioxide
400
5E+00
5E+00
5E+00
Pharyngeal
SWCNT
400
3E-01
5E-01
5E-01
1E+01
1E+01
5E+01
7E+01
1E+01
3.4.4
Genotoxicity of nano-objects in vivo
An important question is, whether carcinogenicity of nano-objects can be predicted by studies on
genotoxicity in vitro. In vitro studies are not subject of the PaFtox database. However, in some in vivo
studies endpoints indicating genotoxicity have been investigated, such as 8-OHdG or HPRT mutations (Tab.
22). As Tab. 22 shows, only few studies are available. Parameter / Effects were found in 33 to 100 % of the
studies for silicon dioxide, Carbon Black, titanium dioxide and SWCNT indicating the potential for
37
genotoxicity also for „inert“ particles. Genotoxicity has been found also in other recent in vivo studies
(Rittinghausen et al. 2012) with a high correlation with cell proliferation. Thus genotoxicity may arise as
primary effect but more probably as secondary effect following cell proliferation.
Tab. 22:
Genotoxic effects (measured and percentage positive effects)
Category time
point
Number of studies effect measured
Percentage positive effects
(positive/measured)
10
10
36
99
189
365
730
36
99
189
365
730
Whole body inhalation - Lung
8-OHdG
Carbon Black
3
HPRT mutations
Carbon Black
3
Silicon dioxide
1
Carbon Black
3
PAH-derived DNA adducts
3
3
67
3
67
67
33
33
33
Whole body inhalation - BALF
HPRT mutations
Carbon Black
6
5
5
67
60
60
Intratracheal instillation - Lung
8-OHdG
Carbon Black
Silicon dioxide
2
3
100
2
SWCNT
100
100
1
8-oxoGua
Silicon dioxide
HPRT mutations
Carbon Black
2
50
Silicon dioxide
2
100
Titanium dioxide
2
50
p53 mutations
Silicon dioxide
5
100
5
6
100
5
40
50
40
Intratracheal instillation - BALF
HPRT mutations
Carbon Black
2
50
Silicon dioxide
2
100
Titanium dioxide
2
0
Traffic light code used for illustration lower limit: 10 percentile set to red and upper limit: 90 percentile set to green
38
Tab. 23:
‘‘LOELS’’ for positive genotoxic effects
Category time point
Target / Organ
Parameter / Effect
10
36
99
189
365
730
Substance
Whole body inhalation
Lung
8-OHdG
Carbon Black
1.4
HPRT mutations
Carbon Black
1.3
1.4
9.4
9.4
Silicon dioxide
BALF
PAH-derived DNA
adducts
Carbon Black
3.1
HPRT mutations
Carbon Black
1.4
1.4
1.4
Intratracheal instillation
Lung
8-OHdG
Carbon Black
9.7
Silicon dioxide
0.8
0.8
SWCNT
BALF
11.3
8-oxoGua
Silicon dioxide
0.75
HPRT mutations
Carbon Black
100
Silicon dioxide
10
Titanium dioxide
100
0.75
3
p53 mutations
Silicon dioxide
6
HPRT mutations
Carbon Black
100
Silicon dioxide
10
Titanium dioxide
-
3.4.5
Carcinogenicity
Fig 5 shows an analysis for the chain of effects: inflammation, hyperplasia, metaplasia and tumour. The
effects were counted for the lowest dose and the earliest time point the effect was observed for all studies.
This graphic gives an indication what effect can be detected at which time point. While inflammation and
hyperplasia were observed within one or three months, metaplasia, cysts, adenoma, carcinoma were
determined at later stages, after one or two years. Similar observations were seen for intratracheal studies
(data not shown).
39
Fig 5:
Number of effects per category time point in whole body inhalation studies on carcinogenicity (query counted
effects at minimum dose and earliest time point)
40
Fig 6 shows the LOELs for the same effects (inflammation, hyperplasia, metaplasia and tumour) for two or
three different particles sizes of Carbon Black, silicon dioxide and titan dioxide. According to this
preliminary analysis, considering the relevance of the effects (inverse the chain of effects) and the
corresponding LOELs, the toxicity could be ranked as follows CB 50 ~ TiO2 500 > SiO2 50 > TiO2 50 >
CB 100 > CB 500 > SiO2 8000. However, the data are limited to only few data points and do not allow any
conclusion regarding the carcinogenicity of nano-objects.
Fig 6:
Effect --- ‘‘LOELs’’ per particle in whole body inhalation studies on carcinogenicity (query counted effects at min
dose and first time point)
3.5 Correlations
3.5.1
Cell counts
Changed counts of different types of lymphocytes are often observed in BALF (Tab. 12). They are related to
(acute) inflammation in the respiratory tract. In the PaFtox database they account for about 15% of all effects
and are mainly determined in BALF (Tab. 24). Frequently affected cell types are alveolar macrophages and
neutrophils. Neutrophils are also called polymorphonuclear neutrophils (PMN). And sometimes the term
PMN is synonymously used to polymorphonuclear leukocytes (PML), which covers in addition to
neutrophils, also eosinophil and basophil granulocytes. In the PaFtox database, both terms are separately
recorded to keep the possibility to identify possible differences (Tab. 24). In the statistical analyses (Tab. 24
41
and Tab. 25), they are separately counted but have been combined at a later stage for effect level/power
analyses.
In Tab. 25 it was investigated, if the effects in BALF occur early or late during treatment. No consistent time
pattern emerges for either of the parameters investigated. No consistent pattern was found in sensitivity
concerning absolute or relative cell counts. Sometimes relative counts were positive more frequently,
sometimes absolute counts.
Tab. 24:
Investigation of cells in different targets (statistically significant increased/measured or investigated)
Parameter / Effect
BALF
relative
58/73
total
75/127
Eosinophils total
3/8
Erythrocytes
Blood
Lung
Nose
0/2
1/10
Granulocytes
0/4
1/1
Leukocytes total
23/26
1/1
Lymphocytes relative
28/48
Lymphocytes total
32/60
87/119
Neutrophils total
72/86
PMN relative
87/123
PMN total
84/130
1/3
12/19
7/23
1/1
Bold marked effects were analysed in more detail.
42
1/3
Tab. 25:
Frequency of increased / measured cell counts in BALF per time point category
Positive / Measured
Category time point
1
3
10
21
36
99
189
365
4/4
4/4
2/2
1/4
2/4
1/2
730
Nose / head only
Alveolar macrophages total
1/1
Lymphocytes total
Neutrophils total
2/2
PMN relative
PMN total
2/5
2/2
1/2
1/1
6/6
6/6
2/3
4 / 10
8 / 12
8 / 12
2/3
12 / 16
12 / 14
4/6
7/7
4/8
8 / 16
5/9
13 / 19
Whole body
Alveolar macrophages relative
/1
Granulocytes
4/4
Leukocytes total
/1
2/2
3/4
4/6
7/9
2/2
7/8
Lymphocytes total
2/2
3/4
/2
1/1
10 / 14
14 / 19
8 / 11
7/8
1/1
2/2
4/4
3/4
3/5
0/1
0/2
11 / 12
7/7
1/3
5/7
1/1
0/1
2/2
1/1
4/5
Neutrophils total
PMN relative
PMN total
1/1
Intratracheal
Alveolar macrophages relative
6/7
4/4
4/4
1/1
2/4
1/3
2/4
7 / 10
7/8
3/6
7 / 11
4/5
0/2
1/2
1/1
0/3
1/1
0/1
4/4
8/8
5/5
Eosinophils total
Leukocytes total
5/6
3/3
1/1
4/4
4/4
1/1
3/4
6/6
Lymphocytes total
1/1
3/6
4/5
1/1
2/9
2/2
1/1
12 / 14
16 / 17
6 / 14
3/3
3/6
4/6
1/1
Neutrophils total
3/3
15 / 18
5/6
3/3
6/7
8/9
1/2
PMN relative
13 / 15
2/2
12 / 17
17 / 22
13 / 16
2/2
1/1
PMN total
16 / 18
15 / 19
11 / 13
9 / 15
1/1
3/3
Pharyngeal
0/3
3/3
4/4
3/3
2/3
Lymphocytes total
2/3
3/3
3/3
3/3
1/3
Neutrophils total
3/3
3/3
3/3
1/1
2/3
PMN total
3/5
1/1
43
5/6
Based on this overview, the effect levels of relative PMNs and relative neutrophils combined were further
investigated in dependency of different measures of dose. As was shown by Oberdörster at al. 2005 for
TiO2, surface area may be a suitable dose measure for comparing effects of nanoparticles with particles of
larger size. Therefore we compared both: dose as mass unit in mg/kg bw for intratracheal studies or mg/m3
for inhalation studies and surface area (dose as mass multiplied by the specific surface if provided for the
respective particles in the studies) for the time point day 1. Fig 7 shows selected trend analyses performed for
all corresponding particles available for the particular application route and time point category. Further
differences in substance, size and crystal structure are marked by different symbols. Fig 7 A shows relative
neutrophils (incl. PMNs) for intratracheal instillations 1 day after instillation in dependency of applied dose
in mg/kg bw. The Plot Fig 7 B shows the same values in dependency of particle surface. Fig 7 C and Fig 7 D
show the corresponding values for the time point categories 5-10 and 11-36 days after instillations,
respectively. Finally, Fig 7 E and Fig 7 F show the results for whole body exposure studies for the time point
categories 37-99 and 100-189 days, respectively. Due to the larger spread in the figure, particle surface as
dose measure seems to better illustrate the dose-response for different particle sizes and compositions.
Therefore the following analyses have been performed solely based on surface. Additionally, a regression
line has been introduced into the figures as visual aid for distinguishing effect levels for different particles.
Overall, a rough dose response relationship can be found for the different particles depending on particle
surface as dose measure.
Some particles (aluminium trioxide or titanium dioxide) lie below the regression line indicating a lower
toxicity than average, while silicon dioxide lies above. Interestingly, this includes also amorphous silicon
dioxide particles which are generally considered to have a lower toxicity than crystalline silica.
Generally the pattern is similar with all durations and also in intratracheal studies compared to inhalation
studies. The steepness of the dose-response curve is also similar. In contrast to studies with intratracheal
instillation few inhalation studies are available for silicon dioxide. Rutile, the crystalline form of titanium
dioxide with higher toxicity lies above the regression line.
44
Fig 7:
Correlation of relative number of neutrophils with particle mass or particle surface area
45
3.5.2
Similar analyses were performed for the relative alveolar macrophage counts in whole body studies and total
alveolar macrophage counts for intratracheal studies (data not shown). A general negative trend was found,
but some particles showed a positive trend: cerium oxide (4 points) and nickel oxide (2 points) in
intratracheal studies at time category 11-36 days after instillation (not at time point 3 days) and titanium
oxide (2 points) in whole body studies at time category 37-99 and 100-189 days (negative in intratracheal
studies).
3.5.3
Total Protein and lactate dehydrogenase
As earlier mentioned (Tab. 12), the effects total protein and lactate dehydrogenase (LDH) in BALF are more
frequently measured than other effects. Therefore, a detailed analysis was performed also for these
endpoints. Tab. 26 shows the corresponding time related distribution of the number of measurements for
both effects.
Tab. 26:
Time related distribution of measurements of total protein and lactate dehydrogenase
Category time point
1
3
10
36
99
189
365
730
1
1
34
29
13
23
1
33
31
15
27
Total protein
Total protein
32
23
21
27
28
2
6
50
11
45
26
37
2
6
Based on this overview (Tab. 26), the percentage changes of total protein measured in BALF were further
investigated in dependency of particle surface area as dose measures for several time point categories: For
intratracheal instillation the time point categories 1, 11-36 and 37-99 days, and for whole body exposure
inhalation studies the time points 37-99 and 100-189 days (Fig 7).
Fig 8 shows selected trend analyses for percentage change of total protein in dependency of particle surface
area performed for all corresponding particles available for the particular application route and time point
category. Further differences in substance, size and crystal structure are marked by different symbols. Fig 8A
shows the results for intratracheal instillations 1 day after instillation. The Plots Fig 8 B, Fig 8 C and Fig 8 D
show the corresponding values for the time point categories 3 days, 11-36 and 37-99 days after instillation,
respectively. Finally, Fig 8 E and Fig 8 F show the results for whole body exposure studies for the time point
categories 37-99 and 100-189 days, respectively.
Overall, a slight dose response relationship can be found for the different particles with particle surface area
as dose measure.
Unfortunately, the data amount does not allow a more detailed analysis regarding differences between
different particle substances or other properties. However, two results are considered as very interesting:
First, nickel led to very high protein contents in BALF at the three time point categories (1 day, 3 and 11-36
days for i.tr. studies), and second nano quartz seems to be less toxic than amorphous nano silicon dioxide.
Aluminium oxide is again less toxic.
46
Fig 8:
Correlation of total protein with particle surface area
47
Time or application route related differences were not identified. The trend and the effect level (with few
exceptions) are similar with all durations and also in intratracheal studies compared to inhalation studies.
Based on the overview presented above (Tab. 26), the percentage changes of LDH measured in BALF were
also further investigated in dependency of particle surface area as dose measures for several time point
categories: For intratracheal instillation the time point categories 1, 11-36 and 37-99 days, and for whole
body exposure inhalation studies the time points 37-99 and 100-189 days (Fig 9).
Fig 9 shows selected trend analyses for percentage change of LDH in dependency of particle surface area
performed for all corresponding particles available for the particular application route and time point
category. Further differences in substance, size and crystal structure are marked by different symbols. Fig 9
A shows the results for intratracheal instillations 1 day after instillation. The Fig 9 B and Fig 9 C show the
corresponding values for the time point categories 5-10 days and 37-99 days after instillation, respectively.
And the plots Fig 9 C, Fig 9 E and Fig 9 F show the results for whole body exposure studies for the time
point categories 37-99, 100-189 and 366-730 days, respectively.
The results differ for each panel.
A slight dose response relationship can be found for the different particles in intratracheal studies at time
point categories 1 day or 5-10 days after instillation and even more powerful correlations can be found for
some substances (cerium oxide, titanium oxide (nano and fine) and Carbon Black) one day after instillation.
The data at time point category 37-99 does not allow analogue conclusions. The analyses for whole body
exposure studies suffer from the limited number of data. The corresponding analyses cover mainly titanium
dioxide and Carbon Black and show only weak correlations (Fig 9 D to F).
Surprisingly, titanium dioxide led to significant higher effect level than cerium oxide one day after
intratracheal instillation (Fig 9 A) and at time point category 5-10 days similar levels were achieved.
However, the slight trend is similar at all time point categories and also in intratracheal studies compared to
inhalation studies.
48
Fig 9:
Correlation of LDH with particle surface area
49
3.5.4
Infiltration
Infiltration of inflammatory cells is the first response of the immune system. The precise description of this
effect differs between the several publications. Currently two types of infiltrations are distinguished in the
database. Terms like hypercellularity, infiltration of (inflammatory) cells, neutrophils, mononuclear cells
(mainly lymphocytes, monocytes and plasma cells) and infiltration of polymorphonuclear leukocytes are
summarised under the effect “infiltration”. Beside this common term, the term “macrophage infiltration” is
separately recorded.
Tab. 27:
Number of studies positive infiltrations
Parameter / Effect
Category time point
1
3
10
36
99
189
365
730
Nose/head only inhalation
Infiltration
2
MWCNT
2
1
Silicon dioxide
2
2
1
1
2
MWCNT
1
Carbon Black
2
Infiltration
Nickel hydroxide
2
1
4
4
6
1
1
Silicon dioxide
5
Silver
1
Titanium dioxide
3
2
1
1
Carbon Black
3
4
1
6
Silicon dioxide
2
1
1
Infiltration
Carbon Black
Iron(II,III) oxide
1
1
MWCNT
Nickel
1
Nickel oxide
Silicon dioxide
2
2
1
1
1
1
1
1
1
1
1
2
1
3
3
10
9
1
1
5
3
1
Cerium(IV) oxide
1
Silicon dioxide
1
1
1
1
1
Titanium dioxide
5
6
5
3
1
Traffic light code used for illustration lower limit: 1 set to red and ideal minimal upper limit: 20 set to green
50
1
1
SWCNT
Titanium dioxide
1
2
Tab. 28:
Parameter /
Effect
‘‘LOELs’’ for infiltration and macrophage infiltration
Category time point
1
3
10
36
99
189
365
730
Aluminum
oxyhydroxide
5.1E+00
MWCNT
Infiltration
Macrophage
infiltration
5.1E+00
8.0E-02
Silicon dioxide
6.6E-01
Aluminum
oxyhydroxide
5.1E+00
MWCNT
1.1E+00
2.9E-01
5.1E+00
1.8E-02
Carbon Black
1.3E+00
Nickel hydroxide
Infiltration
Macrophage
infiltration
2.5E+00
1.2E+00
1.2E+00
2.3E-01
2.3E-01
1.0E+01
1.9E-02
Silicon dioxide
2.3E-01
Silver
8.6E-03
Titanium dioxide
4.0E+00
4.3E+01
4.3E+01
4.3E+01
Carbon Black
1.2E+00
1.2E+00
1.2E+00
1.2E+00
Silicon dioxide
9.0E+00
9.0E+00
9.0E+00
Carbon Black
Iron(II,III) oxide
3.0E+01
1.0E+00
MWCNT
Nickel
5.3E+00
Nickel oxide
Silicon dioxide
8.2E-01
Titanium dioxide
1.0E+00
4.0E-02
4.0E-02
4.0E-02
5.3E+00
5.3E+00
5.3E+00
3.3E-01
3.3E-01
3.3E-01
3.3E-01
3.3E-01
8.2E-03
8.2E-01
8.2E-01
1.0E+00
5.0E+00
1.5E+01
1.1E+01
5.0E+00
5.0E+00
5.0E+00
Cerium(IV) oxide
Macrophage
infiltration
2.5E+01
1.0E+00
SWCNT
Infiltration
9.0E+01
3.5E+00
Silicon dioxide
5.0E+00
5.0E+00
5.0E+00
5.0E+00
5.0E+00
Titanium dioxide
5.0E+00
5.0E+00
3.3E+00
5.0E+00
5.0E+00
LOELs normalised doses for inhalation studies in mg/m3 and cumulative doses in mg/kg bw for instillation studies. Traffic light code used for
3.5.5
Lung weight
Based on our experience with RepDose, we know that lung weight is a sensible term for lung damages. In
contrast to chemicals it might be possible that the lung weight gain resulted from lung burden by the nanoobjects. Therefore, the percentage of burden of the lung weight was determined (see Tab. 29). In all studies
lung burden contributed to a small degree to the lung weight increase, the maximum was 11 % in a
51
carcinogenicity study with titanium dioxide. Therefore, the lung weight gain compared to control is caused
by other factors, e.g. influx of inflammatory cells, or fibrosis.
Tab. 29:
Minimum and Maximum percentages of burden of the lung weight
Category time
point
1-10
min
11-36
max
37-99
100-189
218-365
min
max
min
max
min
max
0.08
5.96
0.10
5.57
0.003
5.03
0.03
0.08
0.005
0.14
0.61
Silicon dioxide
1E-04
Titanium
dioxide
0.27
366-730
min
max
min
max
0.08
0.02
0.05
0.25
0.82
0.29
0.74
0.18
1.02
3E-03
0
5E-04
4E-04
1E-03
4E-04
7E-04
6.46
0.78
6.40
0.58
6.35
0.65
10.88
0.56
0.56
0.36
0.36
0.01
0.01
Aluminum
oxyhydroxide
MWCNT
Silicon dioxide
0.17
0.37
0.15
0.21
Carbon Black
Carbon Black
MWCNT
0.11
0.19
Silicon dioxide
Green: 10 percentile; red: 90 percentile
52
Tab. 30:
Minimum and maximum of percentage change of lung weight
Category time point
1-10
Min
11-36
Max
37-99
100-189
218-365
366-730
Min
Max
Min
Max
Min
Max
Min
Max
Min
Max
115
117
106
106
108
115
121
176
113
169
127
161
129
192
114
235
137
500
194
517
Silicon dioxide
120
230
110
405
100
430
355
420
Titanium dioxide
147
151
120
239
135
450
132
427
149
149
123
123
165
165
MWCNT
Silicon dioxide
114
150
131
133
Carbon Black
Carbon Black
MWCNT
122
122
Silicon dioxide
Traffic light code used for illustration lower limit: 10 percentile set to green and upper limit: 90 percentile set to red
Tab. 30 shows that for all particles and MWCNT lung weight is increased and the increase can be detected
already at early time points, indicating that lung weight is a sensitive parameter. Lung weight was increased
in 47 studies. The lung weight LOEL was at study LOEL in 85 % of these studies.
53
Tab. 31:
‘‘LOELs’’ for lung weight
Category time point
1-10
Norm
Dose
11-36
Orig
Dose
37-99
100-189
218-365
366-730
Norm
Dose
Orig
Dose
Norm
Dose
Orig
Dose
Norm
Dose
Orig
Dose
Norm
Dose
Orig
Dose
Norm
Dose
Orig
Dose
5
28
5
29
1
3
0.3
1.6
0.1
0.5
0.3
2
2
7
1
2
1
2
1
2
Silicon dioxide
6
31
6
31
6
35
10
59
Titanium dioxide
5
10
5
10
5
10
5
10
75
1
250
5
50
0.5
MWCNT
Silicon dioxide
9
51
9
50
Carbon Black
Carbon Black
MWCNT
1
1
Silicon dioxide
LOELs normalised doses for inhalation studies in mg/m3 and cumulative doses in mg/kg bw for instillation studies; * Original dose in mg; Traffic
light code used for illustration lower limit: 10 percentile set to green and upper limit: 90 percentile set to red.
4
Discussion
4.1
Results
4.1.1
Carcinogenicity
The original focus of this project was to derive structure activity relationships for nano-objects with respect
to carcinogenicity. However, as discussed with UBA, this requires a huge data set including numerous
carcinogenicity studies, which are currently not available. Carcinogenicity studies are, however, limited to 4
in inhalation studies (Titanium dioxide – 21 & 250 nm and Carbon Black – 14 & 37 nm) and 20 in
intratracheal studies (Aluminium oxide C - 13 nm, Carbon Black - 14 & 95 nm, Silicon dioxide - 14 & 1100
nm, Titanium dioxide - 21, 25 & 200 nm. As Tab. 29 reveals for Titanium dioxide, the tumours were
induced under severe overload conditions (lung weight contains 11% burden). Therefore, it is questionable,
whether they are suitable for risk assessment at all. In addition, there is no negative study available that
would allow identifying relevant precursors for the development of cancer. Thus, the statement that the
potential carcinogenic risk of nanomaterials can currently be assessed only on a case-by-case basis (Becker
et al., 2011) is still true.
4.1.2
Availability of studies for different particles and fibres
The nano-objects to be analysed were selected together with the sponsor and included Carbon Black and
related compounds, titanium dioxide, aluminium oxide, silicon dioxide, heavy metals and their oxides
(silver, nickel, manganese, iron, and cerium), single and multiwall carbon nano tubes. With respect to
numbers of studies in the database, there is a strong imbalance for these nano-objects: numerous studies are
available for titanium dioxide, Carbon Black and silicon dioxide for different particle sizes, application
54
routes and study durations, while for other particles especially the heavy metals only few studies are
available.
According to our literature search more than 100 additional studies are available including several other
compounds or modifications thereof that could be entered into the database.
4.1.3
Besides the chemical composition, the special properties (high specific surface area or specific volume) of
the nano-objects are considered a crucial/elementary cause for hazard differences compared to larger
particles. Therefore the particle characterisation is as important as the determination of the chemical
composition. However particle characterisation itself in the nanoscale dimension is a relatively new area of
research and technical development. Difficulties arise as the values from different measurement techniques
are difficult or hardly to compare. The importance is recognized by international standardisation organisation
which implemented technical committees (TC) to derive standard protocols for characterisation of
nanomaterials (nano-objects and nanostructured materials) as well as the surface chemistry (TC 229
Nanotechnologies and TC 201 Surface chemical analysis, respectively).
Nanoparticles in nanomaterials are usually present as clusters of aggregates and/or agglomerates. While
primary nanoparticles within aggregates are bound by strong chemical bonds (i.e. covalent or ionic bonds),
binding between agglomerates is caused by van der Waals forces, which are much weaker. However, for
inhalation studies nano-objects may be dispersed in air (aerosols) and for instillations in liquids. The
aggregation status differs between liquid suspensions and aerosols, and is affected by many factors, such as
the dispersion technique, dispersion aids, age of dispersion and experimental conditions. Further details see
BAuA research report F2133 (Schaudien et al., 2011).
The size of the nano-objects or aggregates is considered to be relevant for their deposition behaviour in the
lung. The deposition efficiency in the parts pharynx, bronchi and alveoli differs and depends on the particle
size inhaled. Oberdörster et al illustrates these differences (see Fig 10), based on to the predictive
mathematical model (International Commission on Radiological Protection 1994). Despite the deposition,
also the disposition is considered to be fundamentally different from larger particles (Oberdörster et al.,
2005).
Summarising the costs for the several characterisation tests, it is clear why none of the currently available
studies fulfil the ISO guideline recommendations. On the other hand, the degree of characterisation is an
important part for the quality of a publication / study. An addition it might be possible that some
characterisation data are available to later time point, when identity parameters are consolidated.
55
Fig 10:
4.1.4
Predicted fractional deposition of inhaled particles (Oberdörster et al., 2005)
Application routes
Different application routes are used in experimental studies to assess potential inhalation hazard. In
principal, four exposure techniques can be distinguished, the inhalation techniques nose / head only and
whole body and the instillation types intratracheal and pharyngeal. They differ with respect to exposure
condition, to suitability for regulatory purposes, and to costs (Tab. 32). Due to several reasons, these
application techniques could be compared only to a limited extent.
1. While in inhalation studies, the (nano-)objects are actively inhaled, the exposure in instillation studies
occurs passively. Due to these different exposure techniques, the deposition rate is completely different.
In inhalation studies it depends on several physical parameters (Heyder, 2004; International Commission
on Radiological Protection, 1994) and in instillation studies a full deposition can be assumed.
2. The continuity of exposure differs. In inhalation studies the exposure occurs continuously, at least for
certain duration. In last years, exposure durations of 6 h/d and 5 d/wk are commonly used. In contrast,
instillation studies are always event related exposure studies, also if the instillations are repeated daily.
This difference results in different kinetics in the body, e.g. efficacy of protection mechanism, clearance,
transport or distribution kinetic (Oberdörster et al., 2005; Sturm and Hofmann, 2003, 2006).
56
3. Whole body inhalation studies are technically much easier than nose only and traditionally often used.
However, rats do protect themselves by hiding their nose in the fur. In that case the derived LOEL would
be too high. The second issue is that the particles are deposited on the fur and taken up orally during
their grooming (Oberdörster et al., 2002). In that case the systemic distribution of the particles is biased
and possible effects could not be assigned correctly.
4. Instillation studies have some limitations, like the non-physiological rapid delivery of the particles or
fibres, the possible delivery of non-respirable aggregates, and the bypassing of the nose (Driscoll et al.,
2000). Additionally, especially studies with low instillation number, often apply high doses to achieve
similar doses as in inhalation studies. This leads to acute overload situations. However, when particle
deposition was comparable, pulmonary responses to bolus instillation tended to reflect the pulmonary
response to inhalation (Henderson et al., 1995). Nevertheless, intratracheal studies are very useful for
mechanistic considerations or identification of hazard potential.
5. Pharyngeal studies are considered to better simulate the deposition behaviour than intratracheal studies
and avoid the trauma associated with intratracheal instillation (Rao et al., 2003). Impaction of
nanoparticle in the pharynx and subsequently diffusion into the lung is considered as possible
mechanism for the deposition of airborne nano-objects. Pharyngeal application has the same limitations
as other instillation techniques (Shvedova et al., 2005).
57
Tab. 32:
Comparison of application routes
Application route
Nose / head only
Whole body
Intratracheal
Pharyngeal
Type
Inhalation
Inhalation
Instillation
Instillation
Costs
+++
++
+
+
Feasibility (number of
institutes)
+
++
+++
+++
Exposure type
Active inhalation of
aerosols
Active inhalation of
aerosols
Passive infusion of
suspensions
Passive infusion of
suspensions
Exposure regime
Continuous exposure
several h/d
Continuous exposure
several h/d
Event related exposure
Event related exposure
Feature
State of art method
Rodents protect
themselves (nose in
the fur)
Reflects artificial
situations
Is considered to reflect
the impaction of nanoobject in the pharynx
Particle on the fur
could be taken up
orally
Animal stress by
exposure technique
-
-
Insertion of needle or
cannula through
laryngeal opening
+++
++
No complete list
As described above intratracheal studies can generally be seen only as tool for hazard identification. As
demonstrated in Tab. 16, Tab. 20 and Tab. 21 the toxicity ranking for different particles is similar in
intratracheal studies compared to inhalation studies, e.g. effect levels for silicon dioxide are lower than for
Carbon Black or for titanium dioxide. Therefore intratracheal studies may be useful at a screening level for
identifying the general rank of toxicity for different particles or fibres, while inhalation studies then serve for
risk assessment.
4.1.5
Dose measure
An adequate dose is necessary to derive reliable dose response curves. Instead of particle mass as dose
measure, the particle number or particle surface were proposed and discussed (Donaldson and Poland, 2013;
Oberdörster et al., 2005, 2007; Wittmaack, 2007). As particle number is currently seldom available, a
corresponding analysis could not be performed. Therefore, after an initial comparison of dose measures mass
and surface (Fig 7), surface was used as dose measure in our analyses on neutrophils/PMNs (Fig 7), total
protein (Fig 8) and LDH (Fig 9). Particle surface is calculated by multiplying the specific surface of the
particles with the corresponding mass dose and thus combines the normal dose measure mass with the
property specific surface of the primary particle. The figures clearly demonstrate different effect levels for
the different particles, but no consistent dose response was detected for one particle type in most of the
studies. An exception is LDH at day 1 (Fig 9). There regression lines have been inserted for different particle
types. However, the relevance of this finding only for one parameter and one time point is questionable. In
interpreting this lack of dose response, one has to be aware that several studies are combined in these figures,
where particles may differ from study to study as well as treatment of the particles before exposure.
Furthermore, there are studies with rats or mice, which may give different results.
58
The specific surface used for the calculation of surface in Fig 7, Fig 8 and Fig 9 is based on the data for
characterising the primary particles. However, nano-objects tend to aggregate or agglomerate in the exposure
atmosphere or instillation medium and are therefore seldom applied as real nano-object (Fig 3). Furthermore,
several studies have shown that aggregates/agglomerates stay stable in the lung. On the other side, our
analyses have shown that the toxicity of nanoparticles tend to be higher than the toxicity of larger particles.
Therefore it may be possible that the surface of the individual particles is still available for inducing
reactions in the airways. Therefore it might be helpful to identify a relevant property of the secondary
particles (aggregates/agglomerates) and to include this into the dose measure (e.g. MMAD, specific surface
area or volume, biologically available surface area, (Han et al., 2012)).
An additional drawback is that not for all particles specific surfaces are available. For this reason e.g. silver,
the most toxic particle in our LOEL analyses (Tab. 16, Tab. 20, Tab. 21) is missing in the three figures. On
the other side, this presentation of the data allows an easy comparison of effect sizes.
Overall our analyses of dose response have to be considered as a first tier that was possible within the scope
of this pilot project. More detailed analyses are desirable involving all time points, routes, measures of dose
and also other endpoints. Also according to our analyses particle surface seems to be a better dose measure
than particle mass.
4.1.6
Target organs and important effects
As expected, the respiratory tract with lung, nose, trachea, larynx, pharynx, bronchi, parameters in BALF
and lymph nodes are important targets in studies with nano-objects (Fig 4, Tab. 12).
Many different effects are found in histopathological examinations of the respiratory tract, including
infiltration (Tab. 12, Tab. 27, Tab. 28), fibrosis, inflammation, hyperplasia and different types of tumours
(Tab. 12). In addition lung weight turned out to be a sensitive parameter for particle toxicity (Tab. 29 and
Tab. 30). Increased lung weight may be caused by several processes in the lung such as invasion of
inflammatory cells, haemorrhage, proteinosis, cell proliferation, fibrosis, collagen, oedema, granuloma,
hyperplasia or tumours.
An important effect in BALF is increased numbers of neutrophils/PMNs and macrophages. This parameter
has been further investigated in Fig 7. As described in section 3.5.1. Other frequent effects in BALF are total
protein and LDH (Tab. 12), which also have been further explored (Fig 8, Fig 9).
Many studies investigated only effects in the respiratory tract. Therefore effects at other locations than the
respiratory tract are not detected in a query for frequent effects. To detect also studies, where other target
organs are studied we made a specific query (Tab. 13). Indeed, indications for migration of the nano-objects
were found: In several studies besides the lymph nodes also effects in the haematological system were found.
Some studies reveal effects in the pleura, known to be primarily affected by fibres. Effects in the liver
probably come from oral uptake of the nano-objects, either as a result of clearance or from grooming the fur
in whole body inhalation studies (see 4.1.4).
4.1.7
New parameters
In addition to the parameters mentioned above, different cytokines have been investigated. As Tab. 17 and
Tab. 19 show for the example of heavy metals and nanotubes, there is a large variety and few have been
studied in more than 1 study. Cytokines are often determined in in vitro studies, thus they may build a link to
in vitro studies. However, actually no conclusions can be drawn on the relevance of these investigations.
59
It may be interesting to find out, which of the many new endpoints beyond the scope of OECD guideline
studies in studies with particles or fibres are sensitive and also relevant for investigating the mode of action
of particles and fibres.
4.1.8
LOELs
Effect LOELs are suitable tools for comparing endpoints/effects in toxicological studies. They have already
been successfully used in our analyses with RepDose (Batke et al., 2011; Bitsch et al., 2006; Escher et al.,
2010). Therefore we performed corresponding analyses with the PaFtox database.
At first we have compared the toxicity of different nanomaterials. The most toxic compound was silver (Tab.
16, Tab. 20, Tab. 21). High toxicity was also found for MWCNTs confirming the concern for the toxicity of
nanotubes.
Another important question is, if – in general – nanoparticles are more toxic than larger particles with the
same composition. Our analyses with the PaFtox database (Tab. 20, Tab. 21) have indeed shown that based
on LOELs the toxicity of nanoparticles is generally higher than for the corresponding fine particles. Lower
LOELs have been found for different time points for nanosized Carbon Black, silicon dioxide, titanium
dioxide and Nickel oxide in inhalation and in intratracheal studies than for particles with diameters above
250 nm. The ratio of LOELs was calculated if pairs of LOELs for fine and nanoparticles of the same
substance were available (whole body inhalation and intratracheal instillation). The median ratio of LOELs
of nanoparticles (< 56 nm) and larger particles in Tab. 20 is about 18 (n=31). A similar pattern is observed,
when LOELs are compared for different surface categories (Tab. 21). The median ratio here is 5 for LOELs
of nanoparticles (>70 m2/g) and larger particles (n=26).
Although the general trend is obvious, these results have to be treated with care, because some of the LOELs
are not true LOELs, i.e. dose levels, where only marginal effects occur. In the case of particle or fibre
studies, often only one dose level has been used, that had been selected to cause a significant effect. This is
different from OECD guidelines, where three dose levels are, and the lowest dose is intended to provide the
NOEL, the next dose the “real” LOEL. Thus it is not clear, whether these LOELs are real LOELs, as often
the corresponding NOEL is missing (in 47 of 55 with more than 1 dose level), which would allow to identify
the real LOEL. NOELs are available for subacute or subchronic inhalation studies with Silicon dioxide,
Aluminium oxyhydroxide, Titan dioxide and Carbon Black.
In a similar approach Gebel (2012) has analysed tumour rates in carcinogenicity studies with GBS including
talc, toner, coal dust, titanium dioxide, Carbon Black and diesel exhaust particulates. Gebel found, when
comparing carcinogenic potency of GBP of fine particles and nanoparticles a median ratio of 2.26 if the dose
measure was based on particle mass and a ratio of 0.91 when the dose measure was based on surface area of
the particles. While we made a pairwise comparison i.e. compared fine particles with nanoparticles for each
substance, e.g. TiO2, Gebel has pooled potency indices for all different particles.
For a better comparison, for studies where the data basis consists of 3 dose levels and more, dose response
could be modelled, as performed for TiO2 by Dankovic et al. (2007). Suitable parameters would be
parameters that are measured in many studies and are rather sensitive, such as neutrophils/PMNs, total
protein in BALF, or lung weight. For these parameters one may compare the dose levels of nanoparticles and
larger particles for a given effect size and derive a more reliable ratio.
4.1.9
Exposure duration versus time point
Currently, the PaFtox database contains long term studies i.e. the exposure duration in inhalation studies or
observation duration in instillation studies is longer than 20 days. Shorter time points or categories thereof
60
analysed in this database are interim sacrifices. However, there are many other studies available with shorter
study duration and valuable information on effects as already entered but also information on kinetics and
fate of nano-objects in the body. Therefore these studies could supplement the current database, e.g. the
study of Kreyling (2010).
4.1.10
Overload
If one looks at the definition of lung overload with a particle mass of 1-2 mg/g lung = 0.1 – 0.2% of
(Oberdörster et al., 1990), inhalation studies with titanium dioxide and aluminium oxyhydroxide have been
performed under severe overload conditions (Tab. 29). Also with Carbon Black studies overload was
achieved, while studies with MWCNT just reach the border for overload. Studies with silicon dioxide were
mostly below the threshold. In general it is questionable, whether studies with overload conditions are
reasonable for characterizing the toxicity of a nano-object. It is assumed, that under overload situation
unspecific responses occur that have not much to do with realistic exposure situations. However the latter
have to be evaluated for the purpose of risk assessment.
4.1.11
Reversibility of effects
It was analysed if effects appear or disappear during the exposure or post exposure duration (3.4.2).
Interestingly, no effect disappears during the postexposure duration indicating that no effect was in general
reversible. However, it can be assumed, that at least for some effects the grading/scoring may be lower after
postexposure. This has still to be analysed. Furthermore some effects appear firstly in the postexposure
duration. The later appearing effects are mainly associated with chronic inflammation or follow up reaction
(worst case tumour) to long term irritations by the nano-objects. This general effect pattern is different from
studies with chemicals.
4.1.12
Genotoxicity
Genotoxicity is discussed as one possible mode of action for particles (Schins, 2002). However, in vitro
genotoxicity may not be relevant in vivo due to defence mechanisms (Donaldson et al., 2010). In addition
recent analyses have shown, that carcinogenicity is not predicted very well (Roller, 2011). In the PaFtox
database genotoxicity in vivo was only included as endpoint, when it was analysed in corresponding long
term studies (Tab. 22). For the different nano-objects positive as well as negative results were obtained for
endpoints like 8-OHdG, 8-oxoGua, HPRT mutations and p53 mutations. For analysis of genotoxicity in vivo
in more detail, it would be necessary to include specific genotoxicity studies in vivo into the database.
4.2 Concepts for Grouping of nanomaterials
4.2.1
Groups proposed in the literature
Based on the presumption that there are common modes of action for several types of nanomaterials, groups
for different types of nano-objects have been proposed. These groups will be presented in the following
including the compounds assigned to these groups and the rationale for grouping. Based on our analyses of
the data in the PaFtox database these groupings will be evaluated.
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Nano GBP
GBP means Granular Biopersistent Particles with no or little intrinsic chemical toxicity (inert particles
(DFG, 2013)). This grouping of nanoparticles is consistent with the grouping of granular micromaterials
(Greim and Ziegler-Skylakakis, 2007). Roller and Pott (2006) originally introduced the term GBP with a
slightly different definition, i.e. granular biodurable particles without known significant specific toxicity.
According to Gebel (2012) there are also other abbreviations in the literature, i.e. PSP for poorly soluble
particles and PSLT for poorly soluble, low toxicity particles that are specifying the same group of particles.
(Nano)particles assigned to this group are e.g.:
TiO2, BaSO4 (Dankovic et al., 2007), talc, toner, coal dust, Carbon Black, diesel exhaust emissions
(Dankovic et al., 2007; Gebel, 2012; Roller, 2009), zirconium oxide (Packroff, 2011b).
GBP (nano)particles included in the PaFtox Database are Carbon Black, aluminium oxide, aluminium
trioxide, aluminium oxyhydroxide, titanium dioxide and toner (Tab. 11).
These particles are called biodurable or persistent due to the fact that these particles stay in the lung in
inhalation studies and after intratracheal administration and are not readily removed or dissolved. The PaFtox
database contains data on lung burden of the GBP TiO2, Carbon Black and aluminium oxyhydroxide in
inhalation studies. Particle load in the lung is high, demonstrating the biopersistence of these particles.
Typical effects caused by GBP are inflammation, oxidative stress and secondary genotoxicity at the lung as
the target organ after inhalation (DFG, 2013; Greim and Ziegler-Skylakakis, 2007). Accordingly analyses of
the PaFtox database indicate the following important effects: in BALF neutrophils/PMNs, total cells,
macrophages as well as LDH and total protein are increased, the lung weight is increased. Findings in
histopathological examinations include infiltration of macrophages, hyperplasia of alveolar type II cells,
fibrosis and finally tumours.
Generally no NOELs are provided in these proposals for grouping, that would separate particles with little
intrinsic toxicity from particles with higher toxicity. Based on our data the limit for the LOEL in inhalation
studies could be about 0.1 mg/m3 (Tab. 20). Usually LOELs decrease with increasing exposure duration.
This was not evident from our analyses (Tab. 20), which include also short study durations. Therefore, it
may be possible to use this preliminary threshold irrespective of study duration. When considering this value
one has to be aware that the exposure concentration was normalized with respect to exposure duration to 24
hours/day and 7 days/week. This would include aluminium oxyhydroxide, Carbon Black and amorphous
silicon dioxide, while titanium dioxide would be just below the limit. For intratracheal studies, the data are
too inconsistent to derive such a value.
(Nano)particles with specific toxicity
In contrast to the “inert” particles described above, there are particles with specific toxicity, which have a
higher toxicity than GBS.
Crystalline silica usually is considered to belong to this group. An interesting finding in the analyses of
specific effects of silica (Tab. 18) is that nano amorphous or micro crystalline silica cause necrosis in the
nose, bronchi, and lung reflecting the potential of silica to cause severe effects as a consequence of
inflammation.
Another specific subgroup with high toxicity according to DFG (2013) are metal-based particles (presumably
poorly soluble metal compounds). We propose, however, to use the more specific term “heavy metal-based
particles”, as the light metals titanium and aluminium are also metals.
62
Examples for heavy metals belonging to this group are copper (DFG, 2013), cadmium, nickel, cobalt
(Packroff, 2011b).
In the PaFtox database the following heavy metal compounds are included; iron(II,III) oxide, manganese(IV)
oxide, nickel, nickel hydroxide, nickel oxide and silver.
According to DFG (2013), the “toxicity depends largely on the actual compound, i e. type of metal, metal
species and surface characteristics; relevant endpoints are again oxidative stress and inflammation as well as
metal-specific cellular interactions. “
Analyses of the PaFtox database correspondingly show that heavy metal based particles cause effects similar
to GBS, but in addition, some metal specific effects were found (Tab. 17), e.g.
•
increased weight of the adrenal gland and functional disorders of the brain and nervous system,
increased liver weight for manganese oxide,
•
liver hyperplasia, necrosis, vacuolization and weight increase for silver.
In this group of nanoparticles there may be differences in toxicity depending on solubility as stated by DFG
(2013): “Interestingly, some nanoparticles, as shown in the case of copper, appear to be more toxic and
stimulate more intense inflammatory responses than do their water soluble or microscale particle
counterparts, based on the same metal content. This appears not to be due to increased extracellular
solubility, but may be explained by higher intracellular bioavailability after endocytosis and lysosomal
dissolution of the particles, although this still has to be confirmed experimentally”. Similarly, soluble nickel
sulphate nanoparticles were less toxic than less soluble nickel hydroxide nanoparticles in an inhalation study
(Kang et al., 2011).
Analyses of the influence of solubility on the toxicity of nanoparticles were not possible with the PaFtox
database, because data on solubility were not available. However, instead of water solubility, data on
solubility in artificial body fluids, i.e. alveolar fluid, interstitial fluid and stomach fluid are considered as
more relevant and should be measured for particles that have been investigated in toxicological studies in
order to derive correlations.
Soluble (Nano)particles without significant toxicity
Another group that has been proposed are “soluble nanoparticles without significant toxicity” (Packroff,
2011b). Amorphous silicon dioxide belongs to that group.
This is consistent with our analyses showing low toxicity of silicon dioxide nanoparticles in subchronic to
chronic inhalation studies (Tab. 20, Tab. 21) and a recent evaluation indicating low genotoxic/carcinogenic
potential (Rittinghausen et al., 2013).
In contrast, in intratracheal studies low effect concentrations were found especially for acute effects, e.g.
neutrophil count is a sensitive parameter (Fig 7) and serious effects are found also for amorphous silica, e.g.
inflammation, necrosis, spongiosis, histiocytosis or changes in organ structure (Tab. 18). The acute toxicity
is consistent with recent findings of Pavan et al. (2013), where fully amorphous silica showed more
haemolytic activity than crystalline silica. The authors found that particle size and silanols or siloxane
bridges at the surface are the main actors for the haemolytic activity, while crystallinity or free radical
production are no strict predictors. In other in vitro tests (cytotoxic activity, oxidative stress, and
inflammatory response) all amorphous silicas where less toxic than crystalline, but among the amorphous
silicas pyrogenic silicas, irrespective of their size and agglomeration/ aggregation pattern, appeared more
reactive towards cells than the precipitated ones (Gazzano et al., 2012).
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Thus, it may be justified only for long-term studies to speak about low toxicity of amorphous silicon dioxide.
The low toxicity presumably is mainly triggered by lack of accumulation and adaptation in the body in
inhalation studies or dissolution in intratracheal studies.
We are not aware about clear criteria to be categorized as “soluble” nanoparticle. However, measuring
stability of nanoparticles in artificial body fluids (as indicated above) could provide information on the
dissolution behaviour of nanoparticles and borders should be defined to distinguish between high and low
solubility.
Nanotubes (Nanofibres)
Fibre-like nanomaterials are also called HARN, which refers to High Aspect Ratio Nano-objects with the
following specifications according to Tran et al. (2008):
•
Length to diameter aspect ratio greater than 10 to 1
•
Diameter thin enough to pass ciliated airways
•
Length long enough to initiate the onset of e.g. frustrated phagocytosis and other inflammatory
pathways,
•
The nanomaterial must be biopersistent
From experience with asbestos and synthetic mineral fibres there is concern that the shape of the materials is
an important parameter determining toxicity, especially carcinogenicity. Health effects of asbestos comprise
several types of pleural and parenchymal lung disease associated with inhalation of asbestos fibres.
Nanomaterials with fibrous shape therefore have been separated as a group. In fact several studies have
confirmed the concern for carcinogenicity for fibrous nanomaterials, e.g. Takagi et al. (2008).
In the PaFtox database intratracheal and inhalation studies with SWCNT and MWCNT are included. Their
toxicity ranges from low to high, indicating that in addition to shape as parameter determining toxicity
additional factors are necessary.
When analysing the effects in studies with nanotubes in the PaFtox database, effects typical for asbestos-like
fibres could be found such as thickening of the pleura indicating migration of fibres to the pleura and
potential for induction of mesotheliomas, a tumour in the pleura typical for fibre exposure.
It is important to note that the chemical composition and size may be not sufficient to characterise a particle
sufficiently. For silicon dioxide, crystalline and amorphous silicon dioxide are distinguished. Crystalline
silicon dioxide is more toxic and more stable, while amorphous silicon dioxide has high acute toxicity and
low long term toxicity and is soluble to some degree (Rittinghausen et al., 2013). Similarly for titanium
dioxide two crystalline modifications, rutile and anatase are known that differ with respect to their crystal
structure and with respect to toxicity. In addition to crystal structure, surface modifications are discussed to
change the toxicity (Rossi et al., 2010) and are also subject of an ongoing BAuA research project at
Fraunhofer ITEM (F2246).
4.2.2
Grouping of nanomaterials for optimized testing strategies
Nanomaterials fall under the legislation of REACH, where testing requirements are still discussed. It will be
impossible to perform a full testing program for all nanomaterials with all modifications and in all use
scenarios, notwithstanding that modification or use/release scenarios may alter their biological effects.
64
Alternatively, it should be evaluated, if it is possible to assign certain biological effects to specific material
properties (physical properties and chemical and crystal structure) and group nanomaterials based on these
material properties.
According to Nel et al. (2013), a high throughput-platform is required in analogy to the US ToxCast Project
to investigate the bio-physico-chemical interactions at the nano/bio interface in order to make predictions
about the physico-chemical properties of nanomaterials that may lead to pathology or disease outcome in
vivo. In vivo results are used to validate and improve the in vitro high throughput screening and to establish
structure-activity relationships that allow hazard ranking and modelling by an appropriate combination of in
vitro and in vivo testing.
In the following parameters are briefly specified which may be part of a grouping strategy.
Toxicity in vivo
Performing studies in vivo would be the most reliable and toxicologically sound approach for assessing the
risk of nanomaterials. The question is, whether there may be surrogates for chronic inhalation studies which
render testing of nanomaterials more effective:
For in vivo studies study duration, application route, scope of examination may be analysed.
Concerning study duration a short term inhalation test has been proposed by Ma-Hock et al. (2009) based on
comparison of a 5 day inhalation study with a 90 day inhalation study. Rats shall be exposed for 5 days,
6h/day. Examinations shall be performed at day 3 and 21 postexposure. Sensitive parameters that should be
measured in BALF are cell counts, total protein levels, enzyme activities and mediators of inflammatory cell
infiltration (MCP-1, MCP-3, MDC, IL-8, MIP-2) and cell proliferation (M-CSF, osteopontin) as well as
immune modulation (IL-1, IL-6, TNF-α).
Our preliminary analyses support the view that short-term tests with appropriate postexposure periods may
be sufficient for identification of risks of nano-objects. LOELs for short term exposure of nano-objects (i.e.
10 days) are in a similar order of magnitude as LOELs for longer durations up to chronic (Tab. 20, Tab.
21). Sensitive parameters are number of neutrophils and total protein in BALF as well as lung weight.
Concerning exposure route, intratracheal studies are frequently performed instead of inhalation studies,
which are much more expensive. For risk assessment however, inhalation studies are clearly preferable. For
hazard ranking or forming of groups, intratracheal studies seem to be sufficient. As discussed under “GBS”
dose levels may be provided that allow distinguishing GBS from particles with specific toxicity.
Toxicity in vitro
In in vitro studies genotoxicity and generation of reactive oxygen species are frequently investigated with
respect to their general predictive power for in vivo situations.
In vitro DNA damage tests (especially the Comet assay) and in vitro chromosome mutation assays are the
most frequently used genotoxicity tests on nano-objects. According to Roller (2011) nearly all types of
nanomaterials and control dusts used in the in vitro assays showed genotoxic effects in cell cultures (e.g.
CoCr particles, diesel soot, crystalline and amorphous SiO2,TiO2, Carbon Black) but not consistently in all
studies. Roller concluded that there is no clear correlation of the probability of a positive in vitro test with
particle properties. We came to a similar conclusion in an evaluation of the literature in a research project
from the German BAUA (Ziemann et al., 2011). Unfortunately, in most studies nano-objects used were not
well characterized with respect to chemical composition and physicochemical properties. Therefore more
65
research is necessary with well characterized nanomaterial to identify suitable genotoxicity tests and dose
measures for grouping of nano-objects.
According to Rushton et al. (2010) in vitro tests on ROS generation correlate well with inflammation in vivo
(increase of neutrophils after intratracheal instillation), provided that particles are compared based on particle
surface and the steepest slope of the dose response curve as dose metric.
Toxicokinetics
Fate and kinetics may also give a measure for grouping nanomaterials, for example, if the distribution and
mode of action are the same, but the bioavailability is different (due to differences in size). Identical
distribution patterns in tissues could be a criterion for grouping different nanomaterials.
An example would be grouping of nanomaterials in a separate group that can be found in other locations than
the respiratory tract, raising concern for nano-objects specific systemic effects.
Biopersistence is another important criterion for particle and fibre toxicity, because it is assumed that it has
impact on the mode of action and reversibility of effects. If a nanomaterial rapidly dissolves, the substance
could be treated as a conventional chemical. We are not aware about criteria for defining nanomaterials as
persistent or not.
Physicochemical properties
Several physicochemical properties of nanoparticles are currently proposed to be good predictors of toxicity
of nanoparticles. In the following some examples are given from recent studies:
Crystal structure is an important determinant for reactivity of the surface of a nanomaterial. For silicon
dioxide, crystalline and amorphous silicon dioxide are distinguished. Crystalline silicon dioxide is more toxic
and more stable, while amorphous silicon dioxide has high acute toxicity and low long term toxicity and is
soluble to some degree (Rittinghausen et al., 2013). Similarly for titanium dioxide two crystalline
modifications, rutile and anatase are known that differ with respect to their crystal structure and with respect
to toxicity. Jiang et al. (2008) showed that the in vitro-reactivity of different types of TiO2 nanoparticles
depends on the defect site density in the crystal structure. The number of defects per unit surface area was
translated into the activity in producing ROS in vitro. Amorphous TiO2 has much higher surface defect
density compared to anatase and rutile TiO2 since its structure is not periodic. These analyses correlate with
generation of ROS in in vitro systems as well as pulmonary inflammatory response of nanoparticles
determined by number of neutrophils in lung lavage 24 hous after intratracheal instillation of different
nanomaterials (Rushton et al., 2010).
In addition to crystal structure, surface modifications are discussed to change the toxicity (Rossi et al., 2010)
and are also subject of an ongoing BAuA research project at Fraunhofer ITEM (F2246).
The zeta potential provides information concerning the particles surface charge. It is the electrical potential
created between the surface of a particle with its associated ions, and it’s medium. According toCho et al.
(2012) the acute pulmonary inflammogenicity of 15 nanoparticles showed a significant correlation for low
solubility nanoparticles. This correlation found for zeta potential was not applicable for soluble
nanoparticles.
Conduction band energy levels have recently been proposed as promising approach for future screening of
nanomaterials (Zhang et al., 2012). For 24 different metal oxide nanoparticles of the same size it was shown
that the toxicity of nanoparticles was high when the conduction band energy levels of the metal oxides was
66
close to the redox potential in cells which ranges from -4.12 to -4.84eV. Metals with conduction band
energy outside this range were less toxic. Soluble nanoparticles had a higher toxicity than predicted.
Both analyses support the general view that soluble and insoluble nanomaterials have to be distinguished.
Dose measure for grouping of nanoparticles
In the conventional chemical toxicology, researchers generally use the mass as the metric to describe dose. In
the case of nanoparticles, the particle number or the surface area have been discussed. Several recent studies
have confirmed the use of particle surface as generally usable dose measure for insoluble particles (Han et
al., 2012; Jiang et al., 2008; Rushton et al., 2010).
Concerning effect size, the reference values ED10 or ED50 (dose with 10 or 50 % effect) have been used,
LOELs and NOELs (Roller, 2011), or the steeped slope of the dose response curve (Rushton et al., 2010).
Combination of parameters
As already obvious from groupings of nanoparticles currently proposed (see 4.2.1) several parameters are
necessary for specifying a group, i.e. shape, biopersistence and toxicity.
Fig 11: Illustration of grouping of nanoparticles based on material properties and/or biological effects (from Oomen et al. (2013)).
Fig 11 shows nanomaterials grouped according to surface chemistry/activity, material, shape, size, solubility,
release of ions, ADCE (absorption, distribution, corona formation, and elimination), early biological effects
and also nanomaterials not assignable to a group. A given nanomaterial could also belong to more than one
group.
A large number of property combinations should be considered in order to assess the overall hazard of a
single material type.
The PaFtox database would be a useful tool in the future for collecting data on the parameters described
above, selecting suitable properties and validate the groupings.
5
Conclusion and outlook
The amount and quality of cancer studies as well as studies on genotoxicity of nanomaterials are insufficient
to assess the carcinogenicity of nanomaterials. On the other side cell proliferation in the lung as a
consequence of inflammation may be an important precursor of cancer. Therefore repeated dose inhalation
studies or studies with intratracheal instillation were analysed with the purpose to identify effects that may be
67
precursors of carcinogenicity and to analyse dose response for these effects as basis for regulation of
nanomaterials. For getting a better overview and as basis for statistical analyses and analyses of dose
response the data were collected in the relational database PaFtox.
Despite the heterogeneous data available, some general conclusions can be drawn.
Inflammation was detected with all nanomaterials investigated, however, at different dose levels. Sensitive
parameters of nano-object toxicity are lung weight, numbers of neutrophils/PMNs in BALF, as well as total
protein and LDH in BALF. Further histopathological examinations of the overall respiratory tract reveal
numerous effects that are related to inflammation and subsequent cell proliferation.
Nano-objects have consistently a higher toxicity than non-nano-objects. On the other side there are
considerable differences in the toxicity of different materials. From the particles selected for this project,
silver had the highest toxicity.
Although we did not detect a reliable dose response for effects as a function of particle surface, this dose
measure gave a good graphical representation of the toxicity of different particles. While inhalation studies
are preferable for risk assessment, intratracheal studies are suitable for ranking toxicity of different nanoobjects.
We have used our data also to reflect on proposals for grouping of nanomaterials made by different
institutions. Although groups are proposed, the criteria for assigning nanomaterials to these groups are not
well defined, this refers to toxicity as well as solubility. Concerning toxicity a preliminary LOEL of 0.1
mg/m3 (exposure 24 hours/day, 7 days/week) is proposed to distinguish so called “inert particles” (carbon
black, titanium dioxide, silicon dioxide) from particles with specific toxicity, i.e. particles with specific
toxicity have lower LOELs. Our data further support to have nanotubes in a separate group. Currently
interesting new parameters are identified that influence the toxicity of (nano)particles that may help in the
future for better separation of groups.
Currently, only a selection of particles and fibres was analysed by means of the database. In the last years a
huge amount of short term in vivo and in vitro studies with various nanomaterials was performed. At least
some studies are publicly available and the nanomaterial investigated fulfil more criteria of nano-object
characterisation. It would therefore be desirable to analyse all these data along the lines of this project to
identify more criteria for grouping approaches and /or to identify patterns of nanomaterial behaviour. Also
recent studies with a more guideline-like design as undertaken under the OECD sponsorship program should
be included. By specific searches more information for the physico-chemical characterization of the particles
and fibres should be integrated, such as BET surface or solubility. Based on a broader database, additional
and more specific queries should be performed addressing the question of dose response, particle specific
effects, prediction of long term effects (including cancer) from short term studies, influence of surface
changes on particle toxicity etc. Based on such data, criteria for read across and QSAR-like approaches for
nanoparticles could be developed as well as final criteria for grouping of nanomaterials.
68
6
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71
7
Appendix
7.1
Publications in PaFtox database (October 2012)
Author
Year
Publication
Pages
Bai R et al.
2010
Toxicology Letters
288---300
Belinsky SA, et al.
1995
Report HEI-RR-68 Part III
1-25
Bermudez E, et al.
2002
Toxicological Sciences
86-97
Bermudez E, et al.
2004
347-357
Borm PJA, et al.
2005
Toxicol Appl Pharmacol
157-167
Borm PJA, et al.
2005
Toxicology Applied Pharmacology
157-167
Carter JM, et al.
2006
J Occup Environ Med
1265-1278
Chen H-W, et al.
2006
FASEB Journal
2393-2395
Chen H-W, et al.
2006
FASEB Journal
E1732-E1741
Chen Y, et al.
2004
Toxicology Industrial Health
21-27
Chen Z, et al.
2008
Environ Sci Technol
8985-8992,
Support Info
Cho W-S, et al.
2007
Toxicology Letters
24-33
Choi M, et al.
2008
Toxicology Letters
97-101
Creutzenberg O, et al.
1990
J Aerosol Science
S455-S458
Dasenbrock C, et al.
1996
Toxicology Letters
15-21
Driscoll KE, et al.
1996
372 - 380
Driscoll KE, et al.
1997
Carcinogenesis
423-430
Elder A, et al.
2005
614-629
Endoh S, Uchida K
2008
Int Symp risk assessment manufactured nanoparticles
21-30
Ernst H, et al.
2002
Exp Toxic Pathol
109-126
Ferin J, et al.
1991
J Aerosol Medicine
57-68
Ferin J, et al.
1992
Am J Resp Cell Mol Biol
535-542
Gallagher J, et al.
2003
224-231
Gillespie PA, et al.
2010
Nanotoxicology
106-119
Heinrich U, and Fuhst, R
1992
"Abschlussbericht: Untersuchungen zur tumorinduzierenden Wirkung
von inhalierten Dieselmotorabgasen und anderen Teststäuben in der
Mäuselunge. in German"
1992
"Abschlussbericht: Vergleichende Untersuchungen zur Frage der
tumorinduzierenden Wirkung von Diese1motorabgasen in der
Rattenlunge. in German"
Heinrich U, et al.
1995
533-556
Hext PM, et al.
2002
Ann Occup Hyg
191-196
Hyun J-S, et al.
2008
Toxicology Letters
24-28
Inoue K, et al.
2005
Respiratory Research
1-12
Inoue K, et al.
2010
Free Rad Biol Med
924-934
72
Author
Year
Publication
Pages
Ji JH, et al.
2007
857-871
Johnston CJ, et al.
2000
405-413
Kaewamatawong T, et al.
2006
Toxicologic Pathology
958-965
Kim JS, et al.
2011
Safe Health Work
34-38
Kobayashi N, et al.
2009
Toxicology
110-118
Kobayashi N, et al.
2010
Toxicology
143-153
Koike E, et al.
2008
Int J Immunopathol Pharmacol
35-42
Kolling A, et al.
2011
544-554
Lee KP, & Kelly, DP
1992
Fund Appl Toxicol
399-410
Lee KP, & Kelly, DP
1993
Toxicology
205-222
Lee KP, et al.
1985
Exp Mol Pathol
331-343
Lee KP, et al.
1985
179-192
Lee KP, et al.
1986
Environ Research
144-167
Li J et al.
2007
Environ Toxicol
415-421
Ma JY, et al.
2011
Nanotoxicology
312-325
Ma-Hock L, et al.
2009
468-481
Mauderly JL, et al.
1995
Report HEI-RR-68 Part I
1-106
Morimoto Y, et al.
2010
Nanotoxicology
161-176
Muhle H, et al.
1994
In: Mohr U, et al. (Ed.) Toxic and carcinogenic effects of solid particles
in the respiratory tract
29-41
Nikula KJ, et al.
1995
Fund Appl Toxicol
80-94
Nishi K, et al.
2009
1030-1039
Niwa Y, et al.
2008
Circulation Journal
144-149
Oberdörster G, et al.
1990
J Aerosol Science
384-387
1994
Ann Occup Hyg
295-302
1994
Environ Health Perspect
173-179
Ogami A, et al.
2009
812-818
Oszlánczi G, et al.
2010
Ecotox Environ Safety
2004-2009
Park EJ, et al.
2010
Toxicology
65-71
Pauluhn J
2009
152-167
Pauluhn J
2009
Toxicology
140-148
Pauluhn J
2010
Regulatory Toxicology Pharmacology
78-89
Pauluhn J
2010
226-242
Pott F, and Roller M
2005
Eur J Oncol
249-281
Pott F, and Roller M
2005
Eur J Oncol
3606
Randerath K, et al.
1995
Report HEI-RR-68 Part II, NTIS PB96-138623
Rehn B, et al.
2003
73
84-95
Author
Year
Publication
Pages
Reuzel PGJ, et al.
1991
Food Chem Toxicol
341-354
Roller M
2008
Forschungsprojekt F 2083
Roller M, and Pott F
2006
Ann NY Acad Sci
266-280
Rossi EM, et al.
2010
Particle Fibre Toxicology
1-14
Sager TM
2008
Dissertation
1-290
Sager TM and Castranova V
2009
1-12
Sager TM, et al.
2008
1-15
Santhanam P, et al.
2008
Int J Nanotechnol
30-54
Seiler F, et al.
2001
Arch Toxicol
716-719
Shvedova AA, et al.
2005
Am J Physiol Lung Cell Mol Physiol
L698---L708
Shvedova AA, et al.
2008
Am J Physiol Lung Cell Mol Physiol
L552-L565
Sung J, et al.
2008
567-574
Sung J, et al.
2009
452-461
Warheit DB, et al.
1991
Fund Appl Toxicol
590-601
Warheit DB, et al.
1995
Scand J Environ Health
19-21
Warheit DB, et al.
2004
117-125
Sung J, et al.
2008
567-574
Warheit DB, et al.
2007
270-280
Warheit DB, et al.
2007
Toxicology
90-104
Zhang QW, et al.
1998
J Occup Health
171-176
Zhang QW, et al.
1998
J Toxicol Environ Health A
423-438
74
7.2 Data analysis by databases
In this project, a database has been used for the analysis of repeated dose toxicity studies with nano-objects.
It may be questioned, why the PaFtox Database has been used although several databases on nanomaterials
are available already. Therefore in the following the already existing databases are described briefly and then
the special features of the PaFtox Database are discussed.
7.2.1
NanoSafety Cluster
The EU projects addressing all aspects of nanosafety including toxicology, ecotoxicology, exposure
assessment, mechanisms of interaction, risk assessment and standardisation can be found on the
corresponding website - NanoSafety Cluster 1. This initiative helps to maximise the synergies between the
existing FP6 and FP7 projects. Participation in the NanoSafety cluster is voluntary for projects that
commenced prior to April 2009, and is compulsory for nano-EHS projects that have started since April 2009.
The goal is to provide industrial stakeholders and the general public with appropriate knowledge on the risks
of nanomaterials for human health and the environment.
7.2.2
DaNa
DaNa 2 is an umbrella project aiming at collecting and evaluating scientific results in an interdisciplinary
approach with scientists from different research areas, such as human and environmental toxicology,
biology, physics, chemistry, and sociology. Research findings from the field of human and environmental
nanotoxicology which fulfil the criteria 3 are summarised and presented together with material properties and
possible applications for interested laymen and stakeholders. The DaNa project team wishes to provide for
more transparency and to process results of research on nanomaterials and their influence on humans and the
environment in an understandable way.
7.2.3
Database on Research into the Safety of Manufactured Nanomaterials
The OECD Working Party on Manufactured Nanomaterials (WPMN) developed the “Database on Research
into the Safety of Manufactured Nanomaterials 4”, which is an inventory of safety research information on
manufactured (engineered) nanomaterials. It is designed as a global resource (Database), which details
research projects, helps to identify research needs, provides opportunities to identify the similar fields, and
may lead to create new collaboration and networks.
The following information is stored in distinct fields:
•
Project Title; Start date; End date;
•
Project Status (Current; planned; or completed);
1
http://www.nanosafetycluster.eu/
2
http://nanopartikel.info/cms/lang/en/Projekte/dana;jsessionid=E81E1110A9100B389E0F010A2294E942
3
http://nanopartikel.info/cms/lang/en/Wissensbasis/kriterienkatalog
4
http://www.oecd.org/env/ehs/nanosafety/oecddatabaseonresearchintothesafetyofmanufacturednanomaterials.htm
75
•
Country or organisation;
•
Funding information (where available, on approximate total funding; approximate annual funding;
and funding source);
•
Project Summary; Project URL; Related web links;
•
Investigator information: name, research affiliation, contact details;
•
Categorisation by material name, relevance to the safety, research themes, test methods;
•
Overall outcomes and outputs.
NHECD
NHECD 5 is a free access, robust and sustainable web based information system including a knowledge
repository on the impact of nanoparticles on health, safety and the environment. It includes a robust content
management system (CMS) as its backbone, to hold unstructured data (e.g., scientific papers and other
relevant publications). It also includes a mechanism for automatically updating its knowledge repository,
thus enabling the creation of a large and developing collection of published data on environmental and health
effects following exposure to nanoparticles.
NHECD is based on text mining methods and algorithms that make possible the transition from metadata
(such as author names, journals, keywords) to more sophisticated metadata and to additional information
extracted from the scientific papers themselves. These methods and algorithms were implemented to
specifically extract pertinent information from large amount of documents. NHECD created a systematic
domain model of concepts and terms (i.e., a wide set of domain taxonomies) to support the categorization of
published papers and the information extraction process within this project. Up to now around 10,000 open
source articles have been gathered. NHECD is intended for - academics, industry, public institutions and the
general public.
Besides the databases on research projects or databases / information platforms containing different level of
toxicological information, databases on products containing nanomaterial are available. The Danish
Consumer Council and the Danish Ecological Council have created a nano database 6 that will help
consumers to identify more than 1,200 products that may contain nanomaterials. A German database 7 was
developed by BUND containing more than 1000 products available in Germany.
5
http://nhecd.jrc.ec.europa.eu/
6
http://nano.taenk.dk/ (Posted on December 25, 2012)
7
http://www.bund.net/themen_und_projekte/nanotechnologie/nanoproduktdatenbank/
76
7.2.4
Special features of the PaFtox database
As can be seen from the description of the databases above, they are rather general, either related to
collections of research projects or publications. The PaFTox Database in contrast contains all details for
repeated dose toxicity studies (up to livelong exposure) in a very structured way.
There are several advantages in using such a database for toxicological analyses: the structure of the database
forces the user to collect data systematically. Deficiencies in study description and design become obvious
and can be accounted for in the analyses. Different queries allow systematic data mining of large datasets. As
a result effect patterns, LOELs, dose response can be compared for different types of (nano-)objects, for
different studies and sensitive parameters can be identified. Further, the results of the database queries can be
further analysed by statistics and these results can be easily visualized (e.g. Fig 7, Fig 8, Fig 9), which
improves understanding compared with lengthy descriptions in the text or huge tables.
Based on our experiences with RepDose we made several improvements:
We included the scope of examination. This was especially important for studies with particles and fibres,
because currently available studies with (nano-)objects are mostly not according to guidelines and differ
widely in scope investigated; this relates to organs as well as to endpoints investigated. With the scope of
examination, it is possible to query, how often effects have been examined and how often they were positive.
The scope of examination has already been taken into consideration in our analyses in Tab. 24 and Tab. 25.
As the scope of examination is differing from studies according to OECD guidelines the Klimisch code is not
useful for this database and the criteria used in the database RepDose were adapted for the PaFtox database
(see 2.2.3).
In addition for each effect the effect levels or incidences were entered into the database, including
significance. This rendered data entry very time consuming (see example data sheets in the Appendix), but
allowed queries on effect levels e.g. for neutrophils/PMN levels (Fig 7), total protein (Fig 8), LDH (Fig 9),
lung burden (Tab. 29), lung weight (Tab. 30) and allows future analyses of dose response with the
benchmark dose approach.
7.3
Study reports from PaFtox database (October 2012)
77
Carbon Black
molecular weight 12
g/mol
Study Data
study pk 4108
Specification by Producer / Supplier
object
Primary particle
synonym
Printex 90TM
size
diameter in nm
mean
14
inner diameter in nm
outer diameter in nm
length in nm
SD
min
max
medium
determ. method
distribution type
no data
surface property
cristal structure
shape
specific surface in
m²/g
solubility in mg/l
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
High-purity furnace carbon black
reference
Heinrich & Fuhst (1992)
producer
Degussa, Frankfurt, Germany
Specification by Authors
object
Primary particle
size
diameter in nm
length in nm
mean
SD
min
max
medium
distribution type
determ. method
no data
surface property
cristal structure
shape
specific surface in
m²/g
solubility in mg/l
227
particle density in
g/cm³
additional
1.85
reference
specific volume in
m³/g
at in °C
coarse particles removed by a cyclone with 50% cut-off diameter ca. 1 µm for a flow rate of 100
m3/h; spec. surface area by BETg, 0,04% extractable organic matter
Heinrich & Fuhst (1992)
Hydrodynamic diameter
median
640 nm
distibution type
GSD
2.6
bulk density
min
28. Sep. 12
nm
isoelectric point
no data
mg/ml
in
Page 1 from 8
Carbon Black
max
nm
medium
10-stage Berner impactor
determ. method
sample treatment
zeta potential
mV
in
peak SPR
nm
in
conductivity
µS/cm in
solubility
mg/L
in
at
ºC
applic. medium
dispersant
additional
Study Design
species
rat
strain
Wistar (Crl:(WI) BR)
sex
female
animal/group
100
route
whole-body
age of animal
7w
exposure in h/d
18
exposure in d/w
5
study dur. in d
730
postexp. dur. in d
182
purity
no. of instillation
frequency
whole-body in 6 or 12 m3 horizontal flow type chambers; nominal concentration of 11,6
mg/m3 as time-weighted average of 7,4 mg/m³ for 4 months + 12,2 mg/m³ for 20 months,
cumulative exposure 102,2 g/m³ x h
exposure
(additional)
dose /
concentration
0
11.63
1 dose study
reliability
Unit
B
confidential
mg/m³
interim sacrifices at d 91, 182, 365, 547, 670, 730 and 910 d
number of animals (control/exposed):
carcinogenicity: 220/100, additionally
histology (serial sacrifice): 80/80
DNA-adducts (d 14, 60, 730): 14/14
lung burden: 66/66
lung clearance: 28/28, measured as clearance of radiolabeled Fe2O3 tracer particles (MMAD 0,35
µm)
BALF only after 730 d
CB burden after digestion of tissue measured by turbidity of particles suspended in 0,01 N NaOH
additional
Reference
author
source
volume
07VAG06
year
institution
Fraunhofer ITEM
author
Muhle H, et al.
volume
Abschlussbericht: Vergleichend Untersuchungen zur
Frage der tumorinduzierenden Wirkung von
Dieselmotorabgasen in der Rattenlunge. Final Report
in German
1992
1-56 plus Append
page
year
In: Mohr U, et al. (Ed.) Toxic and carcinogenic effects
of solid particles in the respiratory tract
1994
29-41
page
source
institution
Fraunhofer ITEM
author
source
J Aerosol Science
volume
21, Suppl 1
year
1990
institution
Fraunhofer ITEM
author
Heinrich U, et al.
source
volume
7
year
1995
institution
Fraunhofer ITEM
28. Sep. 12
page
S455-S458
page
533-556
Page 2 from 8
Carbon Black
Scope
organ
animal/group
necropsy
organ weight histopathology
guideline
body weight
100
lung
100
additional
total lung burden (no lavage); histopatho: H&E stain; alveolar lung clearance of
radiolabeled tracer particles; PAH-DNA adducts by 32P-postlabeling (total lung)
100
additional
nasal and paranasal cavities
100
nose
larynx
trachea
100
BALF
alveolar macrophages
granulocytes
lymphocytes
lactate dehydrogenase (LDH)
hydroxyproline
total protein
β-glucuronidase
additional
both lobes, 5x4ml
Effect data
BALF
effect
sex
female
%
sex
dose
8.3736
level
score
significance
%
female
730
100
n.g.
100
female
730
1700
<0.01
1700
sign. increase of LDH activity (% of control) after 24 mo exposure (Fig.25), effect lower than after 18
exposure + 6 mo p-e (see additional data in Study number 3999)
sex
β-glucuronidase
female
dose
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
0
female
730
100
n.g.
100
11.63
female
730
7200
<0.01
7200
additional
sign. increase of β-glucuronidase activity (% of control) after 24 mo exposure (Fig. 26), effect lower
after 18 mo exposure + 6 mo p-e (see additional data in Study number 3999)
effect
sex
total protein
female
dose
0
11.63
additional
28. Sep. 12
timepoint
transient
0
effect
%
11.63
LOEL
mg/kg
11.63
additional
%
LOEL
study unit
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
significance
%
female
730
level
100
score
n.g.
100
female
730
1100
<0.01
1100
sign. increase of totasl protein (% of control) after 24 mo exposure (Fig. 27), effect comparable to 1
exposure + 6 mo p-e (see additional data in Study number 3999)
Page 3 from 8
Carbon Black
effect
sex
hydroxyproline
%
female
dose
sex
timepoint
transient
8.3736
level
score
significance
%
0
female
730
100
n.g.
100
female
730
310
<0.01
310
sign. increase of free hydroxyproline (% of control) after 24 mo exposure (Fig.28), effect higher than
after 18 mo exposure + 6 mo p-e (see additional data in Study number 3999)
effect
sex
alveolar macrophages total
female
dose
sex
LOEL
study unit
11.63
LOEL
mg/kg
transient
2.64318
timepoint
level
0
female
670
0.29
n.g.
100
score
significance
%
0
female
730
0.95
n.g.
100
11.63
female
670
2.29
n.g.
789.65
11.63
female
730
3.05
n.g.
321.05
additional
increase of number of AM after 22 and 24 mo exposure, effects comparable to 18 mo exposure + 4
mo p-e (see additional data in Study number 3999)
effect
sex
PMN total
female
x10E6/ml
11.63
LOEL
mg/kg
11.63
additional
x10E6/ml
LOEL
study unit
dose
sex
LOEL
study unit
11.63
LOEL
mg/kg
transient
8.3736
timepoint
level
0
n.g.
100
100
0
female
670
score
significance
0
female
730
0
n.g.
11.63
female
670
2.19
n.g.
11.63
female
730
3.14
n.g.
additional
%
increase of number of PMNs after 22 and 24 mo exposure, effects higher than after 18 mo exposur
or 6 mo p-e (see additional data in Study number 3999)
effect
sex
lymphocytes total
female
LOEL
study unit
LOEL
mg/kg
transient
x10E6/ml
additional
no effect: number of lymphocytes (Fig.29)
body weight
effect
sex
weight decreased
gram
female
dose
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
0
female
730
417
n.g.
100
0
female
910
390
n.g.
100
11.63
female
730
325
<0.05
77.93
11.63
female
910
323
<0.05
82.82
additional
sign. decrease of bw from ca. d 400 of exposure throughout d 910
clinical symptoms
28. Sep. 12
Page 4 from 8
Carbon Black
effect
sex
mortality
%
female
dose
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
0
female
730
42
n.g.
100
0
female
912
85
n.g.
100
11.63
female
730
56
<0.05
133.33
11.63
female
912
92
<0.05
108.23
additional
mean lifetime sign. decreased; increase of mortality (% of rats) at termination of exposure and after
p-e
lung
effect
sex
weight
gram
female
dose
sex
timepoint
transient
8.3736
level
score
significance
%
female
91
1.28
n.g.
100
0
female
182
1.55
n.g.
100
0
female
365
1.33
n.g.
100
0
female
547
1.54
n.g.
100
0
female
670
1.34
n.g.
100
0
female
730
1.44
n.g.
100
11.63
female
91
2.24
<0.001
175
11.63
female
182
3.64
<0.001
234.83
11.63
female
365
6.46
<0.001
485.71
11.63
female
547
7.64
<0.001
496.1
11.63
female
670
6.93
<0.001
517.16
11.63
female
730
6.83
<0.001
474.3
sign. increases of lung wet wt. with maximum after 18 mo exposure, slightly reversible within 24 mo
exposure (no data for p-e)
effect
sex
burden
female
dose
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
significance
%
0
female
91
0
n.g.
100
0
female
182
0
n.g.
100
0
female
365
0
n.g.
100
0
female
547
0
n.g.
100
0
female
670
0
n.g.
100
level
score
100
0
female
730
0
n.g.
11.63
female
91
7.56
n.g.
11.63
female
182
19.9
n.g.
11.63
female
365
38
n.g.
11.63
female
547
50.2
n.g.
11.63
female
670
45.2
n.g.
11.63
female
730
43.9
n.g.
additional
28. Sep. 12
11.63
LOEL
mg/kg
0
additional
mg/total lung
LOEL
study unit
increase of total lung burden (no lavage) up to 18 mo exposure, slight increase within 24 mo exposu
(no data for p-e), half-time for clearance of TiO2 burden 550 d; further data for 18 mo exposure + pStudy Number 3999
Page 5 from 8
Carbon Black
effect
sex
alveolar clearance
days
female
dose
sex
timepoint
transient
8.3736
level
score
significance
%
female
91
61
n.g.
100
0
female
365
72
n.g.
100
0
female
547
96
n.g.
100
11.63
female
91
244
<0.01
400
11.63
female
365
368
<0.01
511.11
11.63
female
547
363
<0.01
378.12
sign. increase of clearance time for radiolabeled tracer particles (Fe-59-oxid) from 3 mo exposure up
further increase up to 12-18 mo exposure; further data for 18 mo exposure + p-e in Study Number 3
effect
sex
hyperplasia bronchiolo-alveolar
female
%
sex
dose
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
n.g.
100
n.g.
100
0
female
182
0
female
910
0
11.63
female
182
100
minimal
<0.01
female
910
96
severe
<0.01
11.63
additional
sex
fibrosis
female
dose
0
sign. increase of bronchiolo-alveolar hyperplasia in 20/20 rats at 6 mo exposure, and 96/100 rats aft
24 mo expsoure + 6 mo p-e
effect
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
0
significance
%
n.g.
100
0
female
182
0
female
365
10
minimal
n.g.
0
female
547
5.6
minimal
n.g.
0
female
730
0
0
female
910
4.1
mild
n.g.
n.g.
0
female
910
4.1
minimal
n.g.
11.63
female
182
100
minimal
n.g.
11.63
female
365
52.6
mild
n.g.
11.63
female
365
5.3
medium
n.g.
11.63
female
547
66.7
medium
n.g.
11.63
female
547
33.3
mild
n.g.
11.63
female
730
11.1
minimal
n.g.
11.63
female
730
11.1
severe
n.g.
11.63
female
730
77.8
medium
n.g.
11.63
female
910
30
medium
n.g.
11.63
female
910
2
minimal
n.g.
11.63
female
910
57
mild
n.g.
additional
effect
sex
bronchiolo-alveolar adenoma
female
sex
dose
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
100
0
female
910
0
n.g.
11.63
female
910
13
<0.05
additional
100
time-dependent increase of incidences and severity of interstitial fibrosis at >= 6 mo exposure; effec
reversible in severity at 24 mo exposure + 6 mo p-e
%
28. Sep. 12
11.63
LOEL
mg/kg
0
additional
%
LOEL
study unit
increased incidence of bronchiolo-alveolar adenoma 13/100 vs. 0/217 in controls
Page 6 from 8
Carbon Black
effect
sex
bronchiolo-alveolar
adenocarcinoma
female
%
dose
significance
%
0.46
n.g.
100
11.63
female
910
13
<0.05
female
dose
sex
score
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
8.3736
level
score
significance
%
100
0
female
910
0
n.g.
female
910
4
n.g.
increased incidence of squamous cell carcinoma 4/100 vs. 0/217 in controls
sex
benign cystic keratinizing
squamous-cell tumor
female
dose
2826.1
transient
11.63
effect
11.63
transient
8.3736
significance
%
910
0
n.g.
100
11.63
female
910
20
n.g.
female
dose
level
score
increased incidence of benign cystic keratinizing squamous-cell tumour 20/100 vs. 0/217 in controls
sex
clearance
timepoint
LOEL
mg/kg
female
effect
sex
LOEL
study unit
0
additional
%
level
increased incidence of bronchiolo-alveolar adenocarcinoma 13/100 vs. 1/217 in controls
sex
additional
%
8.3736
910
squamous cell carcinoma
timepoint
transient
female
effect
%
11.63
LOEL
mg/kg
0
additional
sex
LOEL
study unit
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
100
0
female
730
0
n.g.
11.63
female
730
100
n.g.
additional
particle-laden macrophages in lungs of all CB-exposed rats
effect
sex
female
LOEL
study unit
LOEL
mg/kg
transient
n.a.
additional
no effect: no increased level of PAH-derived DNA adducts (32P postlabeling)
lymph node
effect
sex
weight
female
%
dose
0
11.63
additional
28. Sep. 12
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
significance
%
female
730
level
100
score
n.g.
100
female
730
800
n.g.
800
8-fold increase at termination of exposure
Page 7 from 8
Carbon Black
effect
sex
burden
mg/organ
female
dose
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
100
0
female
670
0
n.g.
11.63
female
670
6.72
n.g.
additional
increase of retained particle mass in LALN
nose
effect
sex
squamous cell metaplasia
female
%
dose
significance
%
0
n.g.
100
0
female
547
0
n.g.
100
0
female
730
0
n.g.
100
0
female
910
7.7
n.g.
100
11.63
female
365
27.8
n.g.
11.63
female
547
68.8
n.g.
11.63
female
730
55.6
n.g.
11.63
female
910
61
n.g.
degeneration
female
sex
LOEL
study unit
11.63
timepoint
score
792.2
LOEL
mg/kg
transient
8.3736
significance
%
0
n.g.
100
365
9.5
n.g.
100
547
33.3
n.g.
100
female
730
0
n.g.
100
0
female
910
34.4
n.g.
100
11.63
female
182
30
n.g.
11.63
female
365
100
n.g.
1052.6
11.63
female
547
100
n.g.
300.3
11.63
female
730
88.9
n.g.
female
910
75
n.g.
0
female
182
0
female
0
female
0
11.63
additional
effect
female
dose
level
score
218.02
sign. increase of incidences of degenerative changes in mucus membranes of nasal cavity and
paranasal sinuses after >= 6 mo exposure, effect slightly reversible after 24 mo exposure + 6 mo p-e
sex
inflammation
sex
LOEL
study unit
11.63
timepoint
LOEL
mg/kg
transient
8.3736
level
score
significance
%
0
female
365
4.8
n.g.
100
0
female
547
0
n.g.
100
0
female
730
10
n.g.
100
0
female
910
5.9
n.g.
100
11.63
female
365
22.2
n.g.
462.5
11.63
female
547
50
n.g.
11.63
female
730
55.6
n.g.
556
11.63
female
910
40
n.g.
677.96
additional
28. Sep. 12
level
increased incidences of squamous cell metaplasia in epithelia of nasal cavity and paranasal sinuses
after >= 12 mo exposure, effect not reversible after 24 mo exposure + 6 mo p-e
sex
%
8.3736
365
dose
timepoint
transient
female
effect
%
11.63
LOEL
mg/kg
0
additional
sex
LOEL
study unit
increased incidences of inflammatory changes in mucus membranes of nasal cavity and paranasal
sinuses after >= 12 mo exposure, effect slightly reversible after 24 mo exposure + 6 mo p-e
Page 8 from 8
Carbon Black
molecular weight 12
g/mol
Study Data
study pk 4131
object
Primary particle
synonym
Elftex-12
size
diameter in nm
mean
37
length in nm
SD
min
max
medium
determ. method
distribution type
no data
surface property
cristal structure
specific surface in
m²/g
solubility in mg/l
shape
43
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
reference
Ash M, Ash I (Ed.) Handbook of Fillers, Extenders, and Diluents, 2007, p.
producer
Cabot, Boston, MA
object
Primary particle
size
diameter in nm
length in nm
mean
SD
min
max
medium
distribution type
determ. method
no data
surface property
cristal structure
shape
specific surface in
m²/g
solubility in mg/l
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
reference
median
nm
GSD
min
28. Sep. 12
distibution type
bulk density
nm
isoelectric point
bidispers
mg/ml
in
Page 1 from 26
Carbon Black
max
nm
zeta potential
mV
in
peak SPR
nm
in
determ. method
conductivity
µS/cm in
sample treatment
solubility
mg/L
medium
in
at
ºC
applic. medium
dispersant
bimodal particle size distribution: small-size mode (parallel-floe diffusion battery): mass median
diffusion diameter 100 nm, GSD 2,16, 33% of particle mass; large-size mode (cascade
impactor): MMAD 1950 nm, GSD 1,84, 67% of particle mass
additional
Study Design
species
rat
strain
F344/N
sex
male & female
animal/group
114-118
route
whole-body
age of animal
7.5 w
exposure in h/d
16
exposure in d/w
5
study dur. in d
730
postexp. dur. in d
42
purity
no. of instillation
exposure
(additional)
dose /
concentration
Unit
frequency
whole-body, airflow 425 l/min; nominal conc. 2,5 and 6,5 mg/m3, analytical conc. 2,46+-0,03;
6,55 +-0,06 mg/m3
0
2.46
6.55
1 dose study
reliability
B
confidential
mg/m³
additional
Serial sacrifices (N=3 per sex/group) at d 91, 182, 365, 547, 700, terminal sacrifice about d 772;
histopatho all tp, BALF at d 365, 547 & 700;
nonneoplastic findings (sacrifice, euthanasia, death): 547 d (<= 18 mo), N=32-44 per sex/group;
780 d (18 mo - end of life), N=71-86 per sex/group; squamous cysts (incl. died animals): 730 d (18 24 mo), N=54-77 per sex/group; 780 d (24 mo - end of life), N=0-36
neoplastic findings (except sacrificed betw. mo 3 - 12): 780 d (18 mo - end of life), N=105-109 per
sex/group
genotoxicity: PAH-DNA adducts adducts in left lung (all tp, HD, N=3/sex); PAH-DNA adducts in
alveolar type II cells (d 91, HD, N=5/sex); mutations of K-ras or p53 in lung tumors (N=14
adenocarcinomas, N=3 squamous cell carcinomas, N=1 adenosquamous carcinomas); SCE and
micronuclei in circulating lymphocytes ex vivo (only at d 91, N=3/sex); Hb-adducts in erythrocytes
(only at d 91, N=3/sex)
lung burden (all tp): optical density at 620 nm after homogenization of left lung in saline; burden in
LALN (all tp): digestion of tissue with acid, washed residue of inorganic carbon converted to carbon
dioxide and measured by IR spectrometry
Clearance of radiolabeled carbon black [7Be]CB after single exposures on d 91 & 547 (N=8/sex):
whole-body counting at d 0, 4, 7, 13, 28, 35, 42, 56, 73, 84, 98, 112 & 126 d after exposure
Translocation and sequestration of inhaled fluorescent latex microspheres applied on d 81 & 547:
histopatho and morphometry on d 1, 4, 28 & 90 (N=2/sex) to identify microspheres in single alveolar
macrophages, aggregated macrophages & other locations
Reference
author
Mauderly JL, et al.
volume
source
Report HEI-RR-68 Part I
year
1995
institution
Lovelace Biomed. & Environm. Research Institute, Albuquerque
author
Belinsky SA, et al.
volume
institution
28. Sep. 12
page
source
Report HEI-RR-68 Part III
year
1995
page
1-106
1-25
Page 2 from 26
Carbon Black
author
Randerath K, et al.
volume
source
Report HEI-RR-68 Part II, NTIS PB96-138623
year
1995
page
institution
author
volume
Nikula KJ, et al.
25
institution
Inhalation Toxicology Research Institute
source
year
Fund Appl Toxicol
1995
page
80-94
Scope
organ
animal/group
necropsy
guideline
body weight
115
lung
115
additional
mortality
histopatho (right lobe): H&E stain; lung burden (left lobe after lavage, N=3); PAHDNA adducts by 32P-postlabeling in total lung; PAH-DNA adducts in alveolar type II
cells; immunohistochemistry of p53 protein in lung tumors; mutations of K-ras or
p53 in lung tumors by PCR
115
clinical symptoms
115
lymph node
3
additional
particle burden
3
additional
left lobe, 2x2 ml for f, 2x2,5 ml for m
3
BALF
haematology
additional
genotoxicity: SCE and micronuclei in primary cultures of circulating lymphocytes ex
vivo; Hb-adducts
Effect data
BALF
effect
sex
total protein
male &
female
LOEL
study unit
LOEL
mg/kg
transient
mg/ml
additional
no effect (Mauderly: Tab.E1-E3)
effect
sex
glutathione reductase
male &
female
units/liter (U/l)
dose
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
365
34
n.g.
100
0
male & f
547
20
n.g.
100
0
male & f
700
19
n.g.
100
2.46
male & f
365
33
none
97.05
2.46
male & f
547
32
none
160
2.46
male & f
700
40
<0.05
210.52
6.55
male & f
365
48
none
141.17
6.55
male & f
547
52
none
260
6.55
male & f
700
51
none
268.42
additional
28. Sep. 12
sex
LOEL
study unit
sign. increase at d 700 at LD only, increase at HD not sign. due to high SD (Mauderly: Tab.E1-E3)
Page 3 from 26
Carbon Black
effect
sex
β-glucuronidase
male &
female
units/liter (U/l)
dose
sex
timepoint
transient
1.5744
level
score
significance
%
male & f
365
0.26
n.g.
100
0
male & f
547
0.25
n.g.
100
0
male & f
700
0.09
n.g.
100
2.46
male & f
365
4.13
<0.05
1588.5
2.46
male & f
547
5.25
<0.05
2100
2.46
male & f
700
4.07
<0.05
4522.2
6.55
male & f
365
11.94
<0.05
4592.3
6.55
male & f
547
11.13
<0.05
4452
9.63
none
10700
6.55
male & f
sex
male &
female
units/liter (U/l)
sex
dose
700
sign. increase at LD & HD, increase at HD at d 700 not sign. due to high SD, effects not reversible
(Mauderly: Tab.E1-E3)
effect
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
365
112
n.g.
100
0
male & f
547
86
n.g.
100
0
male & f
700
76
n.g.
100
2.46
male & f
365
368
<0.05
328.57
2.46
male & f
547
280
<0.05
325.58
2.46
male & f
700
335
<0.05
440.78
6.55
male & f
365
653
<0.05
583.03
6.55
male & f
547
553
<0.05
643.02
6.55
male & f
700
535
<0.05
703.94
additional
sign. dose-related increase at all tp, not reversible (Mauderly: Tab.E1-E3)
effect
sex
neutrophils total
male &
female
dose
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
365
14
n.g.
100
0
male & f
547
9
n.g.
100
0
male & f
700
4
n.g.
100
2.46
male & f
365
864
<0.05
6171.4
2.46
male & f
547
865
none
9611.1
2.46
male & f
700
854
none
21350
6.55
male & f
365
1417
<0.05
10121
6.55
male & f
547
1976
<0.05
21956
6.55
male & f
700
943
<0.05
23575
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
x10E3/ml
LOEL
study unit
sign. increase at LD at d 365, at HD at all tp, other increases at LD not sign. due to high SD, max. e
at HD at d 547 (Mauderly: Tab.E1-E3)
Page 4 from 26
Carbon Black
effect
sex
male &
female
x10E3/ml
dose
1.5744
significance
%
365
850
n.g.
100
0
male & f
547
575
n.g.
100
0
male & f
700
660
n.g.
100
2.46
male & f
365
1889
<0.05
222.23
2.46
male & f
547
1259
<0.05
218.95
2.46
male & f
700
726
none
110
6.55
male & f
365
1192
none
6.55
male & f
547
1877
none
326.43
6.55
male & f
700
855
none
129.54
level
score
sign. increase of alveolar macrophages at LD at 365 & 547 d, increases at HD not sign. due to high
increases not dose-related, all doses reversible within d 700 (Mauderly: Tab.E1-E3)
sex
leukocytes
male &
female
dose
timepoint
transient
male & f
effect
x10E3/ml
2.46
LOEL
mg/kg
0
additional
sex
LOEL
study unit
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
365
874
n.g.
100
0
male & f
547
591
n.g.
100
0
male & f
700
664
n.g.
100
2.46
male & f
365
2820
<0.05
322.65
2.46
male & f
547
2149
none
363.62
2.46
male & f
700
1583
<0.05
238.4
6.55
male & f
365
2659
<0.05
304.23
6.55
male & f
547
3899
<0.05
659.72
6.55
male & f
700
1806
<0.05
271.98
additional
sign. increase of total leukocytes, increase of LD at d 547 not sign. due to high SD, effect partly
reversible (Mauderly: Tab.E1-E3)
body weight
28. Sep. 12
Page 5 from 26
Carbon Black
effect
sex
weight decreased
male &
female
gram
dose
2.46
LOEL
mg/kg
transient
1.5744
timepoint
level
0
female
300
272
n.g.
100
0
male
450
485
n.g.
100
0
male
500
485
n.g.
100
0
female
500
324
n.g.
100
0
male
680
405
n.g.
100
0
female
730
296
n.g.
100
2.46
female
300
268
none
98.52
2.46
male
450
480
none
98.96
2.46
male
500
465
<0.05
95.87
2.46
female
500
314
<0.05
96.91
2.46
male
680
385
<0.05
95.06
2.46
female
730
268
<0.05
90.54
6.55
female
300
260
<0.05
95.58
6.55
male
450
440
<0.05
90.72
6.55
female
500
296
<0.05
91.35
6.55
female
500
435
<0.05
134.25
6.55
male
680
355
<0.05
87.65
6.55
female
730
244
<0.05
82.43
additional
sex
LOEL
study unit
score
significance
%
sign. dose-dependent decrease of bw, p<0.05 at HD after ~d300(f)+450(m), at LD after d500 (f+m) (
clinical symptoms
effect
sex
mortality
male &
female
median survival ti
dose
2.46
LOEL
mg/kg
transient
1.5744
timepoint
level
0
male
780
639
0
score
significance
%
n.g.
100
100
female
780
696
n.g.
2.46
male
780
605
<0.05
94.67
2.46
female
780
707
none
101.58
6.55
male
780
599
<0.05
93.74
6.55
female
780
675
<0.05
96.98
additional
28. Sep. 12
sex
LOEL
study unit
sign. increase in LD m and HD m & f, m more sensitive, LOEL f = 6,55 mg/m3
Page 6 from 26
Carbon Black
effect
sex
mortality
male &
female
%
dose
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
16
n.g.
100
547
8
n.g.
100
700
92
n.g.
100
female
700
48
n.g.
100
2.46
male
547
30
n.g.
187.5
2.46
female
547
8
n.g.
100
2.46
male
700
92
n.g.
100
2.46
female
700
42
n.g.
87.5
6.55
male
547
28
n.g.
175
6.55
female
547
14
n.g.
175
6.55
male
700
98
n.g.
106.52
female
700
62
n.g.
129.16
0
male
547
0
female
0
male
0
6.55
additional
level
score
increased mortality in m at LD & HD after 500 d, and in f at HD after 700 d (Fig.1); m more sensitive
LOEL f 6,55 mg/m3
haematology
effect
sex
SCE
male &
female
LOEL
study unit
LOEL
mg/kg
transient
n.a.
additional
no effect: no increase of SCE in primary cultures of circulating lymphocytes ex vivo at d 91 (not
investigated at later tp
effect
sex
micronuclei
male &
female
LOEL
study unit
LOEL
mg/kg
transient
n.a.
additional
no effect: no increase of micronuclei in in primary cultures of circulating lymphocytes ex vivo at d 91
investigated at later tp
lung
effect
sex
tumor supressor protein p53
male &
female
n.a.
sex
dose
6.55
timepoint
0
male & f
780
6.55
male & f
780
additional
28. Sep. 12
LOEL
study unit
LOEL
mg/kg
transient
4.192
level
score
no change
significance
%
n.a.
n.a.
p53 protein in 1/3 squamous cell carcinomas in HD group (sex not specified) with 26-50% of nuclei
showing immunoreactivity
Page 7 from 26
Carbon Black
effect
sex
fibrosis
male &
female
%
dose
1.5744
significance
%
547
3
n.g.
100
0
female
547
0
n.g.
100
0
male
772
1
n.g.
100
0
female
772
2
n.g.
100
2.46
male
547
61
n.g.
2033.3
2.46
female
547
52
n.g.
2.46
male
772
92
n.g.
9200
2.46
female
772
96
n.g.
4800
6.55
male
547
75
n.g.
2500
6.55
female
547
78
n.g.
6.55
male
772
99
n.g.
9900
female
772
100
n.g.
5000
level
score
increase of incidences of interstitial fibrosis in alveolar septa for rats died or euthanized < d 547 or >
547 with dose- and time-dependent increase of severity scores, prominent lesion >=365d, correlatin
with increased interstitial aggregation of macrophages; n of control, LD, HD: <547d, m N=32, 44, 44
N=23, 21, 27 ; >547d, m N=86, 71, 71, f N=91, 95, 87 (tab.5,6)
effect
sex
bronchiolo-alveolar
adenocarcinoma
female
dose
timepoint
transient
male
6.55
%
2.46
LOEL
mg/kg
0
additional
sex
LOEL
study unit
sex
LOEL
study unit
2.46
timepoint
0
male
772
0
LOEL
mg/kg
transient
1.5744
level
significance
%
0.92
score
n.g.
100
female
772
0
n.g.
100
2.46
male
772
0.94
n.g.
102.17
2.46
female
772
5.6
n.g.
6.55
male
772
0.94
n.g.
female
772
19
n.g.
6.55
additional
102.17
dose-dependent increase in f (tab.10), pathogenesis from a continuum that began with alveolar epith
hyperplasia; susceptible rats (>365 d up to d 772; control, LD, HD): m N=109, 106, 106, f N=105, 1
105; biased by low survival rates of m >= 680 d
effect
sex
p53 mutations
male &
female
LOEL
study unit
LOEL
mg/kg
transient
n.a.
additional
no effect: no increase of mutations of p53 in lung tumors
effect
sex
K-ras mutations
male &
female
LOEL
study unit
LOEL
mg/kg
transient
n.a.
additional
no effect: no increase of mutations of K-ras in lung tumors
effect
sex
male &
female
LOEL
study unit
LOEL
mg/kg
transient
n.a.
additional
28. Sep. 12
no effect: no increased level of PAH-derived DNA adducts in total lung tissue (32P postlabeling)
Page 8 from 26
Carbon Black
effect
sex
deposits
male &
female
n.a.
dose
sex
timepoint
transient
1.5744
level
score
significance
male & f
365
no change
n.a.
0
male & f
547
no change
n.a.
0
male & f
772
no change
2.46
male & f
365
2.46
male & f
547
n.a.
2.46
male & f
772
n.a.
6.55
male & f
365
n.a.
6.55
male & f
547
n.a.
male & f
772
n.a.
6.55
sex
infiltration
male &
female
dose
n.a.
n.a.
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
0
male & f
365
no change
n.a.
0
male & f
547
no change
n.a.
0
male & f
772
no change
2.46
male & f
365
2.46
male & f
547
n.a.
2.46
male & f
772
n.a.
6.55
male & f
365
n.a.
6.55
male & f
547
n.a.
male & f
772
n.a.
6.55
additional
%
n.a.
n.a.
increased incidences of infiltration of neutrophils as part of inflammatory response >= 365d (see
inflammation)
effect
sex
adenosquamous carcinoma
male &
female
%
sex
dose
%
increased incidences of cholesterol clefts (by-products of necrosis) as part of inflammatory response
365d (see inflammation)
effect
LOEL
study unit
6.55
timepoint
LOEL
mg/kg
transient
4.192
significance
%
0
male
772
0
n.g.
100
level
score
0
100
female
772
0
n.g.
2.46
male
772
0
n.g.
2.46
female
772
0
n.g.
6.55
male
772
0.94
n.g.
6.55
female
772
0.95
n.g.
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
n.a.
LOEL
study unit
single cases at HD m and f (tab.10); susceptible rats (>365 d up to d 772; control, LD, HD): m N=10
106, 106, f N=105, 107, 105; biased by low survival rates of m >= 680 d
Page 9 from 26
Carbon Black
effect
sex
male &
female
%
timepoint
transient
4.192
sex
significance
%
0
male
772
0.92
n.g.
100
level
score
0
female
772
0
n.g.
100
2.46
male
772
0
n.g.
0
2.46
female
772
0
n.g.
6.55
male
772
1.89
n.g.
6.55
female
772
0.95
n.g.
effect
sex
female
sex
dose
205.43
single cases at HD m & f and at control m (tab.10); susceptible rats (>365 d up to d 772; control, LD
HD): m N=109, 106, 106, f N=105, 107, 105; biased by low survival rates of m >= 680 d
%
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male
772
0.92
n.g.
100
0
female
772
0
n.g.
100
2.46
male
772
0.94
n.g.
102.17
2.46
female
772
1.87
n.g.
6.55
male
772
0
n.g.
6.55
female
772
12.4
n.g.
additional
sex
squamous cysts
male &
female
dose
0
dose-dependent increase in f (tab.10); pathogenesis from a continuum that began with alveolar epith
hyperplasia; n of susceptible rats (>365 d up to d 772; control, LD, HD): m N=109, 106, 106, f N=10
107, 105; biased by low survival rates of m >= 680 d
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male
730
0
n.g.
100
0
female
730
0
n.g.
100
0
male
772
0
n.g.
100
0
female
772
0
n.g.
100
2.46
male
730
0
n.g.
2.46
female
730
1.9
n.g.
2.46
male
772
100
n.g.
2.46
female
772
19.4
n.g.
6.55
male
730
5.4
n.g.
6.55
female
730
7.2
n.g.
6.55
female
772
44.4
n.g.
additional
28. Sep. 12
6.55
LOEL
mg/kg
dose
additional
%
LOEL
study unit
f more sensitive, LOEL m = 6,55 mg/m3: increased incidences of squamous cysts (squamous
epithelium with central keratin accumulation) at d 547-730 in LD & HD f with dose- & time-dependen
increase, only HD in m; >730 d increased effect in f, low survival rate of m ; N of control, LD, HD: 54
730 d, m N=77, 72, 74, f N=56, 54, 69; >730d, m N=9, 1, 0, f N=35, 36, 18 (tab.7)
Page 10 from 26
Carbon Black
effect
sex
squamous metaplasia
%
female
sex
0
male
547
0
female
547
0
male
0
transient
4.192
%
0
n.g.
100
0
n.g.
100
772
0
n.g.
100
female
772
0
n.g.
100
2.46
male
547
2
n.g.
2.46
female
547
0
n.g.
2.46
male
772
1
n.g.
2.46
female
772
6
n.g.
6.55
male
547
2
n.g.
6.55
female
547
0
n.g.
6.55
male
772
3
n.g.
6.55
female
772
24
n.g.
level
score
sign. effect at HD f > 547 for rats died or euthanized < d 547 or > d 547 (tab.5,6); N of control, LD, H
<547 d, m N=32, 44, 44, f N=23, 21, 27 ; >547 d, m N=86, 71, 71, f N=91, 95, 87
sex
fibrosis
male &
female
dose
timepoint
LOEL
mg/kg
significance
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male
547
0
n.g.
100
0
female
547
0
n.g.
100
0
male
772
0
n.g.
100
0
female
772
0
n.g.
100
2.46
male
547
0
n.g.
2.46
female
547
0
n.g.
2.46
male
772
6
n.g.
2.46
female
772
17
n.g.
6.55
male
547
2
n.g.
6.55
female
547
7
n.g.
6.55
male
772
25
n.g.
6.55
female
772
31
n.g.
additional
28. Sep. 12
6.55
dose
additional
%
LOEL
study unit
increase of incidences of focal fibrosis with epithelial hyperplasia (nodular focus of fibrosis compose
dense collagen bundles and small alveolar structures, surreounded by hyperplastic epithelium and
associated neutrophils) for rats died or euthanized < d 547 or > d 547 with dose- and time-depende
increase of severity scores >= d485 in f and d516 in m; f more sensitive with higher incidences and
severity scores; n of control, LD, HD: <547d, m N=32, 44, 44, f N=23, 21, 27 ; >547d, m N=86, 71, 7
N=91, 95, 87 (tab.5,6)
Page 11 from 26
Carbon Black
effect
sex
hyperplasia alveolar type II cells
male &
female
%
sex
dose
1.5744
significance
%
91
0
n.g.
100
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
12
n.g.
100
2.46
male & f
91
67
n.g.
2.46
male & f
182
100
n.g.
2.46
male & f
365
100
n.g.
2.46
male & f
547
100
n.g.
2.46
male & f
700
100
n.g.
2.46
male & f
772
100
n.g.
6.55
male & f
91
100
n.g.
6.55
male & f
182
100
n.g.
6.55
male & f
365
100
n.g.
6.55
male & f
547
100
n.g.
6.55
male & f
700
100
n.g.
6.55
male & f
772
100
n.g.
level
score
833.33
833.33
increase of incidences of alveolar epithelial hyperplasia (increased number of alveolar type II cells) (
affected rats), all rats affected at LD >= d 182 & at HD all tp, not reversible during 42 d p-e with dos
and time-dependent increase of grading (see hyperplasia: grading); serial sacrifices: N=3/sex and g
terminal sacrifice control, LD, HD: N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
sex
alveolar proteinosis
male &
female
dose
timepoint
transient
male & f
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
0
male
547
0
n.g.
100
0
female
547
0
n.g.
100
0
male
772
0
n.g.
100
0
female
772
0
n.g.
100
2.46
male
547
0
n.g.
2.46
female
547
14
n.g.
2.46
male
772
0
n.g.
2.46
female
772
27
n.g.
6.55
male
547
18
n.g.
6.55
female
547
56
n.g.
6.55
male
772
25
n.g.
6.55
female
772
99
n.g.
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
%
LOEL
study unit
level
score
f more sensitive, LOEL m 6,55 mg/m3; increase of incidences of alveolar proteinosis for rats died or
euthanized < d 547 or > d 547, dose- and time-dependent increase of severity scores >=365d; effec
seen in LD & HD f, only HD m and at lower incidence than in f; n of control, LD, HD: < 547d, m N=32
44, f N=23, 21, 27 ; >547d, m N=86, 71, 71, f N=91, 95, 87 (tab.5,6)
Page 12 from 26
Carbon Black
effect
sex
fibrosis
male &
female
%
dose
sex
timepoint
male & f
700
LOEL
mg/kg
transient
1.5744
level
significance
%
0
score
n.g.
100
100
0
male & f
772
0
n.g.
2.46
male & f
700
0
n.g.
2.46
male & f
772
28
n.g.
6.55
male & f
700
67
n.g.
male & f
772
33
n.g.
additional
increase of incidences of focal fibrosis (% affected rats), from d 700 at HD & at d 772 at LD, partly
reversible at HD, for grading see fibrosis: grading; serial sacrifices: N=3/sex and group; terminal sac
control, LD, HD: N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
effect
sex
inflammation
male &
female
dose
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
0
n.g.
100
547
0
n.g.
100
772
1
n.g.
100
female
772
5
n.g.
100
2.46
male
547
9
n.g.
2.46
female
547
24
n.g.
2.46
male
772
14
n.g.
1400
2.46
female
772
34
n.g.
680
6.55
male
547
20
n.g.
6.55
female
547
37
n.g.
6.55
male
772
34
n.g.
3400
female
772
63
n.g.
1260
0
male
547
0
female
0
male
0
6.55
additional
sex
male &
female
%
sex
dose
level
score
increase of incidences of chronic-active inflammation, dose- and time-dependent increase of severit
scores >= 365d, focal aggregates of neutrophils, degenerate inflammatory cells, cell debris and
cholesterol clefts; n of control, LD, HD: <547d, m N=32, 44, 44, f N=23, 21, 27 ; <780d, m N=86, 71
f N=91, 95, 87 (tab.5,6)
effect
LOEL
study unit
2.46
transient
1.5744
significance
%
male
547
0
n.g.
100
0
female
547
0
n.g.
100
0
male
772
2
n.g.
100
0
female
772
9
n.g.
100
2.46
male
547
98
n.g.
2.46
female
547
90
n.g.
2.46
male
772
100
n.g.
5000
2.46
female
772
100
n.g.
1111.1
6.55
male
547
100
n.g.
6.55
female
547
93
n.g.
6.55
male
772
100
n.g.
5000
female
772
100
n.g.
1111.1
6.55
timepoint
LOEL
mg/kg
0
additional
28. Sep. 12
2.46
0
6.55
%
LOEL
study unit
level
score
increase of incidences of alveolar epithelial hyperplasia (increased number of alveolar type II cells) f
rats died or euthanized < d 547 or > d 547, dose- and time-dependent increase of severity scores >
d91, localization mainly in centriacinar region; n of control, LD, HD: <547 d, m N=32, 44, 44, f N=23
27 ; <780 d, m N=86, 71, 71, f N=91, 95, 87 (tab.5,6)
Page 13 from 26
Carbon Black
effect
sex
macrophage infiltration
male &
female
%
dose
timepoint
transient
1.5744
significance
%
male
547
3
n.g.
100
0
female
547
0
n.g.
100
0
male
772
0
n.g.
100
0
female
772
4
n.g.
100
2.46
male
547
100
n.g.
3333.3
2.46
female
547
100
n.g.
2500
2.46
male
772
100
n.g.
3333.3
2.46
female
772
100
n.g.
2500
6.55
male
547
10
n.g.
333.33
6.55
female
547
96
n.g.
6.55
male
772
100
n.g.
female
772
100
n.g.
6.55
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
sex
LOEL
study unit
level
score
2500
increase of incidences of enlarged alveolar macrophages (alveolar macrophage hyperplasia) for rats
died or euthanized < d 547 or > d 547; at later tps macrophage aggregation in centriacinar region; n
control, LD, HD: <547 d, m N=32, 44, 44, f N=23, 21, 27 ; >547d, m N=86, 71, 71, f N=91, 95, 87
Page 14 from 26
Carbon Black
effect
sex
burden
male &
female
mg/total lung
dose
2.46
LOEL
mg/kg
transient
1.5744
timepoint
level
91
0
n.g.
100
female
91
0
n.g.
100
male
182
0
n.g.
100
0
female
182
0
n.g.
100
0
male
365
0
n.g.
100
0
female
365
0
n.g.
100
0
male
547
0
n.g.
100
0
female
547
0
n.g.
100
0
male
700
0
n.g.
100
0
female
700
0
n.g.
100
2.46
male
91
1.7
n.g.
100
2.46
female
91
1.7
n.g.
2.46
male
182
5.6
n.g.
2.46
female
182
3.3
n.g.
2.46
male
365
7.9
n.g.
2.46
female
365
6.2
n.g.
2.46
male
547
16
n.g.
2.46
female
547
12.1
n.g.
2.46
male
700
24.7
n.g.
2.46
female
700
17.3
n.g.
6.55
male
91
5.9
n.g.
6.55
female
91
4.9
n.g.
6.55
male
182
13.7
n.g.
6.55
female
182
11
n.g.
6.55
male
365
15.1
n.g.
6.55
female
365
12.2
n.g.
6.55
male
547
29.9
n.g.
6.55
female
547
22.7
n.g.
6.55
male
700
40.1
n.g.
6.55
female
700
36.9
n.g.
0
male
0
0
additional
28. Sep. 12
sex
LOEL
study unit
score
significance
%
347.05
dose & time-dependent increase of total lung burden (no lavage), progressive accumulation accelera
after 365d control values assumed, tab.3)
Page 15 from 26
Carbon Black
effect
sex
metaplasia
male &
female
%
dose
sex
LOEL
mg/kg
transient
1.5744
significance
%
0
n.g.
100
547
0
n.g.
100
780
0
n.g.
100
female
780
1
n.g.
100
2.46
male
547
2
n.g.
2.46
female
547
29
n.g.
2.46
male
780
15
n.g.
2.46
female
780
66
n.g.
6.55
male
547
25
n.g.
6.55
female
547
52
n.g.
6.55
male
780
66
n.g.
female
780
97
n.g.
547
0
female
0
male
0
sex
metaplasia
male &
female
dose
level
score
6600
9700
increase of incidences of bronchiolar-alveolar metaplasia, dose- and time-dependent increase of
severity scores >= 365d for rats died or euthanized < d 547 or > d 547, f more sensitive with higher
incidences and severity scores; n of control, LD, HD: <547d, m N=32, 44, 44, f N=23, 21, 27 ; <780d
N=86, 71, 71, f N=91, 95, 87 (tab.5,6)
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
3
n.g.
100
2.46
male & f
365
0
n.g.
2.46
male & f
547
50
n.g.
2.46
male & f
700
83
n.g.
2.46
male & f
772
94
n.g.
6.55
male & f
365
33
n.g.
6.55
level
score
male & f
547
100
n.g.
6.55
male & f
700
100
n.g.
6.55
male & f
772
100
n.g.
additional
sex
alveolar clearance
male &
female
dose
3133.3
3333.3
dose- and time-dependent increase of incidences of bronchiolar-alveolar metaplasia (% affected rat
from d 365 at HD % d 547 at LD, not reversible, for grading see bronchiolar-alveolar metaplasia:
grading; serial sacrifices: N=3/sex and group; terminal sacrifice control, LD, HD: N=34, 18, 12
(Mauderley: Tab.F1, F3, F5, F7, F8, F9)
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
0
male & f
217
79
n.g.
100
level
score
0
male & f
673
69
n.g.
100
2.46
male & f
217
41
n.g.
51.89
2.46
male & f
673
18
n.g.
26.08
6.55
male & f
217
24
n.g.
30.37
male & f
673
14
n.g.
20.28
6.55
additional
28. Sep. 12
timepoint
male
6.55
%
2.46
0
additional
%
LOEL
study unit
decrease of alveolar clearance rate indicated from decreased clearance of radiolabeled tracer partic
([7Be]CB) on d 126 each after inhalative uptake on d 91 or d 547 = tps of d 217 & d 673 (100% %retained, calculated from Tab.11)
Page 16 from 26
Carbon Black
effect
sex
clearance
male &
female
n.a.
dose
sex
1.5744
level
score
significance
91
no change
n.a.
0
male & f
182
no change
n.a.
0
male & f
365
no change
n.a.
0
male & f
547
no change
n.a.
0
male & f
772
no change
2.46
male & f
91
2.46
male & f
182
n.a.
2.46
male & f
365
n.a.
2.46
male & f
547
n.a.
2.46
male & f
772
n.a.
6.55
male & f
91
n.a.
6.55
male & f
182
n.a.
6.55
male & f
365
n.a.
6.55
male & f
547
n.a.
6.55
male & f
772
n.a.
macrophage damage
male &
female
dose
sex
0
male & f
n.a.
n.a.
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
547
no change
no change
significance
n.a.
0
male & f
772
male & f
547
2.46
male & f
772
n.a.
6.55
male & f
547
n.a.
6.55
male & f
772
n.a.
n.a.
increase of cellular debris from macrophages at >= 547 d
effect
sex
macrophages interstitial
male &
female
dose
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
0
male & f
91
no change
n.a.
0
male & f
182
no change
n.a.
0
male & f
365
no change
n.a.
0
male & f
547
no change
n.a.
0
male & f
772
no change
2.46
male & f
365
2.46
male & f
547
n.a.
2.46
male & f
772
n.a.
6.55
male & f
91
n.a.
6.55
male & f
182
n.a.
6.55
male & f
365
n.a.
6.55
male & f
547
n.a.
male & f
772
n.a.
6.55
additional
%
n.a.
2.46
additional
%
particle-laden macrophages seen at all tps, however, at later tps free particles increased due to cellu
damage of macrophages
sex
28. Sep. 12
timepoint
transient
male & f
effect
n.a.
2.46
LOEL
mg/kg
0
additional
n.a.
LOEL
study unit
%
n.a.
n.a.
few particle-containing interstitial macrophages at d 91 at HD, marked increase at >=d 365 at both d
including both single and aggregated macrophages
Page 17 from 26
Carbon Black
effect
sex
foci
male &
female
n.a.
dose
0
sex
male & f
level
547
score
no change
significance
547
n.a.
547
n.a.
foci with increased amount of epithelial hyperplasia including some foci with papillary projections of
hyperplasitc epithelium or multilayering of hyperplastic epithelial cells at d 547
male &
female
dose
0
6.55
additional
sex
LOEL
study unit
6.55
timepoint
LOEL
mg/kg
transient
4.192
level
score
significance
male & f
91
6.1
n.g.
male & f
91
20.1
<0.05
sex
squamous cysts
male &
female
sex
LOEL
study unit
2.46
timepoint
0
male & f
700
LOEL
mg/kg
1.5744
level
significance
%
0
score
n.g.
100
100
0
male & f
772
0
n.g.
male & f
700
0
n.g.
2.46
male & f
772
28
n.g.
6.55
male & f
700
33
n.g.
6.55
male & f
772
42
n.g.
increase of incidences of squamous cysts (% affected rats) from d 700 at HD & at d 772 at LD, not
reversible; serial sacrifices: N=3/sex and group; terminal sacrifice control, LD, HD: N=34, 18, 12
effect
sex
squamous metaplasia
male &
female
dose
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
0
male & f
772
0
n.g.
2.46
male & f
772
0.11
n.g.
6.55
male & f
772
0.75
n.g.
additional
329.5
transient
2.46
additional
%
100
sign. increase of PAH-derived DNA adducts in alveolar type II cells at HD at d 91 (no data for LD or
other tp)
effect
dose
%
n.a.
male & f
28. Sep. 12
1.5744
male & f
sex
grading
timepoint
transient
6.55
effect
%
2.46
LOEL
mg/kg
2.46
additional
adducts/10E9 bas
LOEL
study unit
%
100
increase of grading (% affected lung) of alveolar squamous metaplasia at d 772 (for incidences see:
squamous metaplasia: %), grading scale (% of affected lung): 1 (<10%), 2 (10-25%), 3 (25-50%), 4
(>50%); serial sacrifices: N=3/sex and group; terminal sacrifice control, LD, HD: N=34, 18, 12
Page 18 from 26
Carbon Black
effect
sex
male &
female
grading
dose
sex
timepoint
transient
1.5744
level
score
significance
%
male & f
91
0
n.g.
100
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
0.06
n.g.
100
2.46
male & f
91
1
n.g.
2.46
male & f
182
1.33
n.g.
2.46
male & f
365
2
n.g.
2.46
male & f
547
2.33
n.g.
2.46
male & f
700
3
n.g.
2.46
male & f
772
2.69
n.g.
6.55
male & f
91
2
n.g.
6.55
male & f
182
2.17
n.g.
6.55
male & f
365
3
n.g.
6.55
male & f
547
3
n.g.
6.55
male & f
700
3.5
n.g.
6.55
male & f
772
3.42
n.g.
sex
metaplasia
male &
female
dose
4483.3
5700
dose- and time-dependent increase of grading of enlarged alveolar macrophages (alveolar macroph
hyperplasia), for incidences see macrophage infiltration: %; serial sacrifices: N=3/sex and group;
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
0.06
n.g.
100
2.46
male & f
365
0
n.g.
2.46
male & f
547
1
n.g.
2.46
male & f
700
1.33
n.g.
2.46
male & f
772
2
n.g.
6.55
male & f
365
0.5
n.g.
6.55
male & f
547
1.83
n.g.
6.55
male & f
700
2.5
n.g.
6.55
male & f
772
3.08
n.g.
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
grading
LOEL
study unit
3333.3
5133.3
dose- and time-dependent increase of grading (% affected lung) of bronchiolar-alveolar metaplasia f
d 365 at HD & d 547 at LD, not reversible (for incidences see: bronchiolar-alveolar metaplasia: %),
grading scale (% of affected lung): 1 (<10%), 2 (10-25%), 3 (25-50%), 4 (>50%); serial sacrifices:
N=3/sex and group; terminal sacrifice control, LD, HD: N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7
F9)
Page 19 from 26
Carbon Black
effect
sex
male &
female
grading
sex
dose
1.5744
significance
%
91
0
n.g.
100
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
0.18
n.g.
100
2.46
male & f
182
1.33
n.g.
2.46
male & f
365
2
n.g.
2.46
male & f
547
2.17
n.g.
2.46
male & f
700
3
n.g.
2.46
male & f
772
3.06
n.g.
6.55
male & f
91
2
n.g.
6.55
male & f
182
2
n.g.
6.55
male & f
365
2.83
n.g.
6.55
male & f
547
2.83
n.g.
6.55
male & f
700
3.17
n.g.
6.55
male & f
772
3.67
n.g.
level
score
1700
2038.9
dose- and time-dependent increase of grading (% affected lung) of alveolar epithelial hyperplasia; no
reversible during 42 d p-e (for incidences see: hyperplasia: %), grading scale (% of affected lung): 1
(<10%), 2 (10-25%), 3 (25-50%), 4 (>50%); serial sacrifices: N=3/sex and group; terminal sacrifice
sex
male &
female
dose
timepoint
transient
male & f
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
100
0
male & f
772
0
n.g.
2.46
male & f
365
0.17
n.g.
2.46
male & f
547
0.33
n.g.
2.46
male & f
700
0.33
n.g.
2.46
male & f
772
0.78
n.g.
6.55
male & f
365
1.33
n.g.
6.55
male & f
547
1.33
n.g.
6.55
male & f
700
1.83
n.g.
6.55
male & f
772
3.5
n.g.
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
grading
LOEL
study unit
dose- and time-dependent increase of grading (% affected lung) of alveolar proteinosis from d 365,
reversible (for incidences see: alveolar proteinosis: %), grading scale (% of affected lung): 1 (<10%)
(10-25%), 3 (25-50%), 4 (>50%); serial sacrifices: N=3/sex and group; terminal sacrifice control, LD
N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
Page 20 from 26
Carbon Black
effect
sex
male &
female
%
dose
significance
%
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
0
n.g.
100
2.46
male & f
365
17
n.g.
2.46
male & f
547
33
n.g.
2.46
male & f
700
33
n.g.
2.46
male & f
772
56
n.g.
6.55
male & f
365
100
n.g.
6.55
male & f
547
67
n.g.
6.55
male & f
700
67
n.g.
level
score
male & f
772
100
n.g.
dose- and time-dependent increase of incidences of alveolar proteinosis (% affected rats) from d 36
not reversible, for grading see alveolar proteinosis: grading; serial sacrifices: N=3/sex and group;
effect
sex
fibrosis
male &
female
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
700
0
n.g.
100
100
0
male & f
772
0
n.g.
2.46
male & f
700
0
n.g.
2.46
male & f
772
0.5
n.g.
6.55
male & f
700
1.33
n.g.
male & f
772
0.75
n.g.
6.55
additional
28. Sep. 12
1.5744
365
dose
timepoint
transient
male & f
6.55
grading
2.46
LOEL
mg/kg
0
additional
sex
LOEL
study unit
increase of grading (% affected lung) of focal fibrosis from d 700 at HD & at d 772 at LD, partly reve
at HD (for incidences see: fibrosis: %), grading scale (% of affected lung): 1 (<10%), 2 (10-25%), 3 (
50%), 4 (>50%); serial sacrifices: N=3/sex and group; terminal sacrifice control, LD, HD: N=34, 18,
Page 21 from 26
Carbon Black
effect
sex
fibrosis
male &
female
grading
dose
2.46
timepoint
LOEL
mg/kg
transient
1.5744
level
score
significance
%
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
0.03
n.g.
100
2.46
male & f
182
0
n.g.
2.46
male & f
365
0.33
n.g.
2.46
male & f
547
2
n.g.
2.46
male & f
700
2.83
n.g.
2.46
male & f
772
2.67
n.g.
6.55
male & f
182
0.33
n.g.
6.55
male & f
365
1.17
n.g.
6.55
male & f
547
2.5
n.g.
6.55
male & f
700
3
n.g.
male & f
772
3
n.g.
6.55
additional
28. Sep. 12
sex
LOEL
study unit
8900
10000
dose- and time-dependent increase of grading (% affected lung) of alveolar septal fibrosis; from d 18
HD & d 365 at LD, not reversible (for incidences see: fibrosis: %), grading scale (% of affected lung)
(<10%), 2 (10-25%), 3 (25-50%), 4 (>50%); serial sacrifices: N=3/sex and group; terminal sacrifice
Page 22 from 26
Carbon Black
effect
sex
weight
male &
female
gram
dose
2.46
LOEL
mg/kg
transient
1.5744
timepoint
level
91
1.32
n.g.
100
female
91
0.94
n.g.
100
male
182
1.4
n.g.
100
0
female
182
1.03
n.g.
100
0
male
365
1.58
n.g.
100
0
female
365
1.09
n.g.
100
0
male
547
1.77
n.g.
100
0
female
547
1.45
n.g.
100
0
male
700
1.99
n.g.
100
0
female
700
1.25
n.g.
100
2.46
male
91
1.35
none
102.27
2.46
female
91
1.06
none
112.76
2.46
male
182
1.71
none
122.14
2.46
female
182
1.17
<0.05
113.59
2.46
male
365
2.17
<0.05
137.34
2.46
female
365
1.59
<0.05
145.87
2.46
male
547
2.69
none
151.97
2.46
female
547
2.01
none
138.62
2.46
male
700
3.12
none
156.78
2.46
female
700
2.42
<0.05
193.6
6.55
male
91
1.56
none
118.18
6.55
female
91
1.21
<0.05
128.72
6.55
male
182
2.12
none
151.42
6.55
female
182
1.68
<0.05
163.1
6.55
male
365
3.31
<0.05
209.49
6.55
female
365
2.56
<0.05
234.86
6.55
male
547
3.5
<0.05
197.74
6.55
female
547
3.71
<0.05
255.86
6.55
male
700
4.48
none
225.12
6.55
female
700
4.95
<0.05
396
0
male
0
0
additional
28. Sep. 12
sex
LOEL
study unit
score
significance
%
dose- & time-dependent increase of lung wt, sign. in f HD, f more sensitive: sign. effect seen earlier
and at more tp at LD (tab.4)
Page 23 from 26
Carbon Black
effect
sex
male &
female
%
dose
sex
timepoint
transient
1.5744
significance
%
male & f
91
0
n.g.
100
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
6
n.g.
100
2.46
male & f
91
100
n.g.
2.46
male & f
182
100
n.g.
2.46
male & f
365
100
n.g.
2.46
male & f
547
100
n.g.
2.46
male & f
700
100
n.g.
2.46
male & f
772
100
n.g.
6.55
male & f
91
100
n.g.
6.55
level
score
male & f
182
100
n.g.
6.55
male & f
365
100
n.g.
6.55
male & f
547
100
n.g.
6.55
male & f
700
100
n.g.
6.55
male & f
772
100
n.g.
sex
fibrosis
male &
female
dose
1666.7
1666.7
increase of incidences of enlarged alveolar macrophages (alveolar macrophage hyperplasia), all rat
affected from d 91-772; for grading see macrophage infiltration: grading; serial sacrifices: N=3/sex a
group; terminal sacrifice control, LD, HD: N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
3
n.g.
100
2.46
male & f
182
0
n.g.
2.46
male & f
365
33
n.g.
2.46
male & f
547
100
n.g.
2.46
male & f
700
100
n.g.
2.46
male & f
772
100
n.g.
6.55
male & f
182
33
n.g.
6.55
male & f
365
67
n.g.
6.55
male & f
547
100
n.g.
6.55
male & f
700
100
n.g.
6.55
male & f
772
100
n.g.
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
%
LOEL
study unit
level
score
3333.3
3333.3
increase of incidences of alveolar septal fibrosis (% affected rats), from d 182 at HD & d 365 at LD,
rats affected at >= d 547, not reversible, for grading see fibrosis: grading; serial sacrifices: N=3/sex
group; terminal sacrifice control, LD, HD: N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
Page 24 from 26
Carbon Black
effect
sex
inflammation
male &
female
grading
dose
sex
timepoint
transient
1.5744
level
score
significance
%
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
0.09
n.g.
100
2.46
male & f
182
0.17
n.g.
2.46
male & f
365
0.17
n.g.
2.46
male & f
547
1.17
n.g.
2.46
male & f
700
0.67
n.g.
2.46
male & f
772
1
n.g.
6.55
male & f
182
0
n.g.
6.55
male & f
365
0.17
n.g.
6.55
male & f
547
2
n.g.
6.55
male & f
700
1.83
n.g.
male & f
772
1.75
n.g.
6.55
sex
inflammation
male &
female
dose
1111.1
1944.4
dose- and time-dependent increase of grading (% affected lung) of chronic active inflammation; max
d 547, partially reversible thereafter (for incidences see: inflammation: %), grading scale (% of affec
lung): 1 (<10%), 2 (10-25%), 3 (25-50%), 4 (>50%); serial sacrifices: N=3/sex and group; terminal
sacrifice control, LD, HD: N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
effect
sex
LOEL
study unit
2.46
timepoint
LOEL
mg/kg
transient
1.5744
significance
%
0
male & f
182
0
n.g.
100
0
male & f
365
0
n.g.
100
0
male & f
547
0
n.g.
100
0
male & f
700
0
n.g.
100
0
male & f
772
9
n.g.
100
2.46
male & f
182
17
n.g.
2.46
level
score
male & f
365
17
n.g.
2.46
male & f
547
100
n.g.
2.46
male & f
700
100
n.g.
2.46
male & f
772
72
n.g.
6.55
male & f
182
0
n.g.
6.55
male & f
365
17
n.g.
6.55
male & f
547
100
n.g.
6.55
male & f
700
100
n.g.
6.55
male & f
772
100
n.g.
additional
28. Sep. 12
2.46
LOEL
mg/kg
0
additional
%
LOEL
study unit
800
1111.1
increase of incidences of chronic active inflammation (% affected rats), single rats on d 182 & 365, a
rats affected at >= d 547, partially reversible at LD after 42 d p-e, for grading see inflammation: grad
serial sacrifices: N=3/sex and group; terminal sacrifice control, LD, HD: N=34, 18, 12 (Mauderley:
Tab.F1, F3, F5, F7, F8, F9)
Page 25 from 26
Carbon Black
effect
sex
squamous metaplasia
male &
female
%
dose
sex
LOEL
study unit
2.46
timepoint
transient
1.5744
significance
%
0
n.g.
100
772
6
n.g.
772
33
n.g.
0
male & f
772
2.46
male & f
6.55
male & f
additional
LOEL
mg/kg
level
score
increase of incidences of alveolar squamous metaplasia (% affected rats) at d 772, for grading see
squamous metaplasia: grading; serial sacrifices: N=3/sex and group; terminal sacrifice control, LD, H
N=34, 18, 12 (Mauderley: Tab.F1, F3, F5, F7, F8, F9)
lymph node
effect
sex
burden
male &
female
mg/organ
dose
2.46
LOEL
mg/kg
transient
1.5744
timepoint
level
91
0
n.g.
100
female
91
0
n.g.
100
male
182
0
n.g.
100
0
female
182
0
n.g.
100
0
male
365
0
n.g.
100
0
female
365
0
n.g.
100
0
male
547
0
n.g.
100
0
female
547
0
n.g.
100
0
male
700
0
n.g.
100
0
female
700
0
n.g.
100
2.46
male
91
0.026
n.g.
2.46
female
91
0.004
n.g.
2.46
male
182
0.37
n.g.
2.46
female
182
0.233
n.g.
2.46
male
365
0.949
n.g.
2.46
female
365
0.763
n.g.
2.46
male
547
2.461
n.g.
2.46
female
547
1.478
n.g.
2.46
male
700
3.124
n.g.
2.46
female
700
1.782
n.g.
6.55
male
91
0.188
n.g.
6.55
female
91
0.173
n.g.
6.55
male
182
0.978
n.g.
6.55
female
182
0.766
n.g.
6.55
male
365
2.934
n.g.
6.55
female
365
1.471
n.g.
6.55
male
547
4.318
n.g.
6.55
female
547
1.997
n.g.
6.55
male
700
4.448
n.g.
female
700
2.076
n.g.
0
male
0
0
6.55
additional
28. Sep. 12
sex
LOEL
study unit
score
significance
%
time-related increase of LALN burden, m > f, control values assumed (Mauderly: Tab.D1-D5)
Page 26 from 26
Titanium dioxide
molecular weight 79.9
g/mol
Study Data
study pk 4099
object
Primary particle
synonym
TiO2 P25
size
diameter in nm
mean
21
length in nm
SD
min
15
max
40
medium
determ. method
distribution type
no data
surface property
cristal structure
Anatase
shape
specific surface in
m²/g
hydrophilic
specific volume in
m³/g
solubility in mg/l
at in °C
particle density in
g/cm³
additional
3.8
reference
MSDS Aug.2011, Degussa A.G., Frankfurt am Main, Germany
producer
Degussa A.G., Frankfurt am Main, Germany
hydrophil pyrogen
object
Primary particle
size
diameter in nm
length in nm
mean
SD
min
max
medium
determ. method
distribution type
no data
surface property
cristal structure
~80 % anatase and ~20 %
rutile
48
shape
specific surface in
m²/g
solubility in mg/l
particle density in
g/cm³
additional
reference
specific volume in
m³/g
at in °C
Titanium dioxide P25, Degussa, Germany; coarse particles removed by a cyclone (shift of MMAD
from 1500 nm to 800 nm)
Heinrich et al. (1995)
median
800 nm
distibution type
GSD
1.8
bulk density
28. Sep. 12
no data
mg/ml
Page 1 from 9
Titanium dioxide
min
nm
isoelectric point
max
nm
zeta potential
medium
10-stage Berner impactor
determ. method
sample treatment
in
mV
in
peak SPR
nm
in
conductivity
µS/cm in
solubility
mg/L
in
at
ºC
applic. medium
dispersant
additional
Study Design
species
rat
strain
Wistar (Crl:(WI) BR)
sex
female
animal/group
100
route
whole-body
age of animal
7w
exposure in h/d
18
exposure in d/w
5
study dur. in d
730
postexp. dur. in d
180
purity
no. of instillation
exposure
(additional)
dose /
concentration
frequency
whole-body in 6 or 12 m3 horizontal flow type chambers; nominal concentration of 10 mg/m3
as time-weighted average of 7,2 mg/m³ for 4 months + 14,8 mg/m³ for 9,5 months + 9,4
mg/m³ for 5,5 months
cumulative exposure 88,1 g/m³ x h
1 dose study
0
10
reliability
Unit
B
confidential
mg/m³
additional
interim sacrifices at d 91, 182, 365, 547, 670, 730 and 910 d
number of animals (control/exposed):
carcinogenicity: 220/100, additionally
histology (serial sacrifice): 80/80
DNA-adducts: 14/14
lung burden: 66/66
lung clearance: 28/28, measured as clearance of radiolabeled Fe2O3 tracer particles (MMAD 0,35
µm)
Ti burden via AAS after ashing of tissue
Reference
author
source
volume
07VAG06
year
institution
Fraunhofer ITEM
author
Muhle H, et al.
volume
Abschlussbericht: Vergleichend Untersuchungen zur
Frage der tumorinduzierenden Wirkung von
Dieselmotorabgasen in der Rattenlunge. Final Report
in German
1992
1-56 plus Append
page
year
In: Mohr U, et al. (Ed.) Toxic and carcinogenic effects
of solid particles in the respiratory tract
1994
29-41
page
source
institution
Fraunhofer ITEM
author
source
J Aerosol Science
volume
21 Suppl 1
year
1990
institution
Fraunhofer ITEM
28. Sep. 12
page
S455-S458
Page 2 from 9
Titanium dioxide
author
Heinrich U, et al.
source
volume
7
year
1995
institution
Fraunhofer ITEM
page
533-556
Scope
animal/group
organ
necropsy
guideline
lung
10
additional
trachea
total lung burden (no lavage), alveolar lung clearance of radiolabeled tracer
particles; histopatho: H&E stain
10
larynx
10
nose
10
additional
body weight
nasal and paranasal cavities
100
BALF
10
lymphocytes
granulocytes
hydroxyproline
total protein
β-glucuronidase
additional
mortality
both lobes, 5x4ml
100
lymph node
additional
10
burden in LALN
Effect data
BALF
effect
sex
x10E6
female
dose
sex
LOEL
study unit
7.2
10
timepoint
level
score
significance
%
female
670
0.29
n.g.
100
0
female
730
0.95
n.g.
100
10
female
670
1.52
n.g.
524.13
10
female
730
1.02
n.g.
107.36
increase of number of AM at 22 and 24 mo of exposure, effect slightly lower than after 18 mo expos
6 mo p-e (addtional BALF data in Study Number 4002)
effect
sex
hydroxyproline
female
10
sex
timepoint
dose
LOEL
study unit
LOEL
mg/kg
transient
7.2
significance
%
0
female
730
100
n.g.
100
10
female
730
260
<0.01
260
additional
28. Sep. 12
transient
0
additional
%
LOEL
mg/kg
level
score
sign. increase of free hydroxyproline at termination of 24 mo exposure, effect higher than after 18 m
exposure + 6 mo p-e (addtional BALF data in Study Number 4002)
Page 3 from 9
Titanium dioxide
effect
sex
total protein
dose
female
10
sex
timepoint
transient
7.2
level
score
significance
%
0
female
730
100
n.g.
100
female
730
890
<0.01
890
effect
sign. increase of total protein at termination of 24 mo exposure, effect lower than after 18 mo exposu
sex
β-glucuronidase
dose
LOEL
study unit
female
10
sex
timepoint
LOEL
mg/kg
transient
7.2
level
score
significance
%
0
female
730
100
n.g.
100
10
female
730
6750
<0.01
6750
additional
sign. increase of β-glucuronidase activity at termination of 24 mo exposure, effect lower than after 1
exposure + 6 mo p-e (addtional BALF data in Study Number 4002)
effect
sex
female
10
%
sex
timepoint
dose
LOEL
study unit
LOEL
mg/kg
transient
7.2
significance
%
0
female
730
100
n.g.
100
10
female
730
1300
<0.01
1300
additional
effect
female
dose
level
score
sign. increase of LDH activity at termination of 24 mo exposure, effect lower than after 18 mo expos
sex
granulocytes
x10E6
LOEL
mg/kg
10
additional
%
LOEL
study unit
sex
LOEL
study unit
LOEL
mg/kg
transient
7.2
10
timepoint
level
score
significance
%
0
female
670
0
n.g.
100
0
female
730
0
n.g.
100
10
female
670
1.42
n.g.
female
730
2.67
n.g.
10
additional
increase of number of granulocytes (10E6/ml) at 22 and 24 mo of exposure, effect higher than after
mo exposure + 6 mo p-e (addtional BALF data in Study Number 4002)
body weight
effect
sex
weight decreased
gram
dose
LOEL
study unit
female
10
sex
timepoint
LOEL
mg/kg
transient
7.2
level
score
significance
%
100
0
female
730
417
n.g.
10
female
730
365
<0.05
additional
87.52
sign. decrease of bw from d 400 until termination of exposure
clinical symptoms
28. Sep. 12
Page 4 from 9
Titanium dioxide
effect
sex
mortality
%
female
dose
sex
LOEL
study unit
LOEL
mg/kg
transient
7.2
10
timepoint
level
score
significance
%
0
female
730
42
n.g.
100
0
female
910
85
n.g.
100
10
female
730
60
<0.05
142.85
10
female
910
90
<0.05
105.88
additional
sign. decease of mean lifetime; mortality increased at termination of exposure an after 6 mo p-e
lung
effect
sex
weight
gram
female
dose
sex
transient
7.2
10
timepoint
level
score
significance
%
female
91
1.28
n.g.
100
0
female
182
1.55
n.g.
100
0
female
365
1.33
n.g.
100
0
female
547
1.54
n.g.
100
0
female
670
1.34
n.g.
100
0
female
730
1.44
n.g.
100
10
female
91
1.93
<0.001
150.78
10
female
182
2.96
<0.001
190.96
10
female
365
4.48
<0.001
336.84
10
female
547
6.16
<0.001
400
10
female
670
5.72
<0.001
426.86
10
female
730
5.29
<0.001
367.36
sign. increase of lung burden from 3 mo throughout 24 mo of exposure, maximum level at 18 mo
exposure, partly reversible up to 24 mo exposure (no data for p-e)
effect
sex
burden
female
dose
sex
LOEL
study unit
10
LOEL
mg/kg
transient
7.2
significance
%
0
female
91
0
n.g.
100
0
female
182
0
n.g.
100
0
female
365
0
n.g.
100
0
female
547
0
n.g.
100
0
female
670
0
n.g.
100
timepoint
level
score
100
0
female
730
0
n.g.
10
female
91
5.2
n.g.
10
female
182
23.2
n.g.
10
female
365
34.8
n.g.
10
female
547
40
n.g.
10
female
670
37.7
n.g.
10
female
730
39.3
n.g.
additional
28. Sep. 12
LOEL
mg/kg
0
additional
mg/organ
LOEL
study unit
increase of total lung burden (no lavage) from 3 mo throughout 24 mo exposure, maximum level at 1
mo exposure, practically not reversible up to 24 mo exposure (no data for p-e); clearance half-time
d; further data for 18 mo exposure + p-e in Study Number 4002
Page 5 from 9
Titanium dioxide
effect
sex
alveolar clearance
days
female
dose
sex
LOEL
study unit
7.2
10
timepoint
level
score
significance
%
female
91
61
n.g.
100
0
female
365
72
n.g.
100
0
female
547
96
n.g.
100
10
female
91
208
<0.01
340.98
10
female
365
403
<0.01
559.72
10
female
547
357
<0.01
371.87
sign. increase of alveolar clearance half- time of radiolabeled tracer particles, maximum at 12 mo
exposure (no data for p-e); further data for 18 mo exposure + p-e in Study Number 4002
effect
sex
hyperplasia bronchiolo-alveolar
female
10
%
sex
timepoint
dose
0
LOEL
study unit
LOEL
mg/kg
transient
7.2
level
score
%
n.g.
100
n.g.
100
182
0
female
910
0
10
female
182
100
medium
n.g.
female
910
99
severe
n.g.
10
increased incidences of bronchiolo-alveolar hyperplasia in all rats of interim sacrifices, and in 99/100
at 24 mo exposure + 6 mo p-e with increase in severity
effect
sex
fibrosis
female
dose
0
significance
female
additional
sex
LOEL
study unit
LOEL
mg/kg
transient
7.2
10
timepoint
level
score
0
significance
%
n.g.
100
0
female
182
0
female
265
10
minimal
n.g.
0
female
547
5.6
minimal
n.g.
0
female
730
0
0
female
910
4.1
minimal
n.g.
n.g.
0
female
910
4.1
minimal
n.g.
10
female
182
100
minimal
n.g.
10
female
365
15
medium
n.g.
10
female
365
75
mild
n.g.
10
female
547
15
severe
n.g.
10
female
547
75
medium
n.g.
10
female
547
10
mild
n.g.
10
female
730
100
medium
n.g.
10
female
910
60
medium
n.g.
10
female
910
36
mild
n.g.
additional
effect
sex
LOEL
study unit
female
10
sex
timepoint
dose
LOEL
mg/kg
transient
7.2
level
score
significance
0
female
910
0
n.g.
10
female
910
4
none
additional
100
sign. increase of incidences and severity of interstitial fibrosis after 6 mo throughout 24 mo exposure
mo p-e, effect partly reversible during p-e
%
28. Sep. 12
transient
0
additional
%
LOEL
mg/kg
%
100
increased incidence of bronchiolo-alveolar adenoma at 24 mo exposure + 6 mo p-e
Page 6 from 9
Titanium dioxide
effect
sex
bronchiolo-alveolar
adenocarcinoma
female
%
dose
sex
10
timepoint
significance
%
n.g.
100
730
0
n.g.
100
910
0.46
n.g.
100
female
547
10
none
10
female
730
11
none
10
female
910
13
<0.05
547
0
female
0
female
10
dose
level
score
LOEL
study unit
female
10
sex
timepoint
LOEL
mg/kg
transient
7.2
level
score
significance
0
female
91
no change
n.a.
0
female
182
no change
n.a.
0
female
365
no change
n.a.
0
female
547
no change
n.a.
0
female
670
no change
n.a.
0
female
730
no change
10
female
91
10
female
182
n.a.
10
female
365
n.a.
10
female
547
n.a.
10
female
670
n.a.
female
730
n.a.
10
additional
%
n.a.
n.a.
accumulation of particle-laden macrophages
effect
sex
female
10
sex
timepoint
dose
2826.1
increased incidences of bronchiolo-alveolar adenocarcinoma at 18 mo and 24 of exposure (2/20 and
1/9) throughout 6 mo p-e (13/100), sign. at termination of study
sex
LOEL
study unit
LOEL
mg/kg
transient
7.2
significance
%
0
female
547
0
n.g.
100
0
female
730
0
n.g.
100
0
female
910
0
n.g.
100
10
female
547
15
none
10
female
730
22
none
10
female
910
3
none
additional
28. Sep. 12
transient
0
female
effect
%
LOEL
mg/kg
7.2
0
additional
n.a.
LOEL
study unit
level
score
increased incidences of squamous cell carcinoma at 18 mo and 24 of exposure (3/20 and 2/9), lowe
incidence at 6 mo p-e (3/100)
Page 7 from 9
Titanium dioxide
effect
sex
benign cystic keratinizing
squamous-cell tumor
female
%
dose
LOEL
study unit
10
sex
LOEL
mg/kg
transient
7.2
timepoint
significance
%
0
n.g.
100
730
0
n.g.
100
910
0
n.g.
100
female
547
10
none
10
female
730
22
none
10
female
910
20
<0.05
0
female
547
0
female
0
female
10
additional
level
score
increased incidences of benign cystic keratinizing squamous-cell tumours at 18 mo and 24 of expos
(2/20 and2/9) throughout 6 mo p-e (20/100), sign. at termination of study
lymph node
effect
sex
burden
mg/organ
dose
LOEL
study unit
female
10
sex
timepoint
LOEL
mg/kg
transient
7.2
level
score
significance
0
female
670
0
n.g.
10
female
670
5.75
n.g.
additional
%
100
burden in LALN
nose
effect
sex
atrophy
%
dose
LOEL
study unit
female
10
sex
timepoint
7.2
level
score
significance
%
100
0
female
730
0
n.g.
female
730
22.2
n.g.
increased incidence of atrophy of respiratory epithelium
effect
sex
squamous cell metaplasia
female
10
sex
timepoint
dose
LOEL
study unit
LOEL
mg/kg
transient
7.2
significance
%
0
female
365
0
n.g.
100
0
female
547
0
n.g.
100
0
female
730
0
n.g.
100
0
female
910
7.7
n.g.
100
10
female
365
5
n.g.
10
female
547
35
n.g.
10
female
730
77.8
n.g.
10
female
910
56
n.g.
additional
28. Sep. 12
transient
10
additional
%
LOEL
mg/kg
level
score
727.27
sign. increase of incidences of squamous cell metaplasia in mucus membranes of nasal cavity and
paranasal sinuses at >= 12 mo exposure, effect partly reversible after 24 mo exposure and 6 mo p-e
Page 8 from 9
Titanium dioxide
effect
sex
inflammation
%
female
dose
sex
transient
7.2
10
timepoint
level
score
significance
%
female
365
4.8
n.g.
100
0
female
547
0
n.g.
100
0
female
730
10
n.g.
100
0
female
910
5.9
n.g.
100
10
female
365
10
n.g.
208.33
10
female
547
35
n.g.
10
female
730
44.4
n.g.
444
10
female
910
30
n.g.
508.47
sign. increase of incidences of inflammative changes in mucus membranes of nasal cavity and
effect
sex
degeneration
female
dose
sex
LOEL
study unit
LOEL
mg/kg
transient
7.2
10
timepoint
significance
%
0
n.g.
100
365
9.5
n.g.
100
547
33.3
n.g.
100
female
730
0
n.g.
100
0
female
910
34.4
n.g.
100
10
female
182
30
n.g.
10
female
365
85
n.g.
894.73
10
female
547
95
n.g.
285.28
10
female
730
100
n.g.
female
910
59
n.g.
0
female
182
0
female
0
female
0
10
additional
28. Sep. 12
LOEL
mg/kg
0
additional
%
LOEL
study unit
level
score
171.51
sign. increase of incidences of degenerative changes in mucus membranes of nasal cavity and
Page 9 from 9
Titanium dioxide
g/mol
Study Data
study pk 4116
object
Primary particle
synonym
TiO2 particle grade
size
diameter in nm
length in nm
mean
SD
min
max
medium
determ. method
distribution type
no data
surface property
cristal structure
rutile
shape
specific surface in
m²/g
solubility in mg/l
spherical
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
Titanium dioxide particle grade
reference
Lee et al., 1985, 1986
producer
du Pont Co., Inc.
object
Primary particle
size
diameter in nm
mean
250
length in nm
SD
min
max
medium
distribution type
assumed
determ. method
no data
surface property
spherical
cristal structure
shape
specific surface in
m²/g
solubility in mg/l
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
refer to pigment-TiO2 --> usually 200-300 nm
reference
median
1600 nm
GSD
min
28. Sep. 12
distibution type
bulk density
nm
isoelectric point
no data
mg/ml
in
Page 1 from 11
Titanium dioxide
max
nm
medium
no data
determ. method
sample treatment
zeta potential
mV
in
peak SPR
nm
in
conductivity
µS/cm in
solubility
mg/L
in
at
ºC
applic. medium
dispersant
Ranges of MMAD 1500, 1700 and 1600 nm at LD, MD and HD with respirable fractions (<
13000 nm MMAD) of 78, 89 and 84%, respectively
additional
Study Design
species
rat
strain
CD
sex
male & female
animal/group
71-79
route
whole-body
age of animal
5w
= 0.99
purity
exposure in h/d
6
exposure in d/w
5
study dur. in d
730
postexp. dur. in d
0
no. of instillation
frequency
exposure
(additional)
dose /
concentration
whole-body exposure chamber 3,85 qm, nominal conc. 0, 10, 50, and 250 mg/m3; analytical
conc. 10,6+- 2,1; 50,3+-8,8; 250,1+- 24,7 mg/m3
0
10.6
50.3
250.1
Unit
mg/m³
1 dose study
reliability
B
confidential
additional groups for interim sacrifices after 91 & 182 d (5/sex and group) and 365 d (10/sex and
group) corresponding to timepoints of 91, 182, 365, 730 d
Ti burden in lung by ICPS after digestion of tissue; burden in other tissues as particle deposition by
microscopy
additional
Reference
author
volume
Lee KP, et al.
41
institution
Du Pont Company, Haskell Laboratory Toxicology & Industrial Medicine
author
Lee KP, et al.
source
Exp Mol Pathol
volume
42
year
1985
institution
author
Lee KP, et al.
source
volume
79
year
1985
institution
source
year
Environ Research
1986
page
144-167
page
331-343
page
179-192
Scope
organ
animal/group
necropsy
guideline
lung
75
additional
trachea
28. Sep. 12
total lung (no lavage); histopatho: H&E stain, PAS, trichrome and silver stain; light
microscopy, TEM and SEM; Ti burden by ICPS
75
Page 2 from 11
Titanium dioxide
nose
75
body weight
100
lymph node
75
additional
burden: dust deposition in tracheobronchial lymph nodes, cervical lymph nodes,
and in mesenteric lymph nodes by light microscopy
75
additional
burden: dust deposition by light microscopy
75
liver
spleen
additional
burden: dust deposition by light microscopy
Effect data
body weight
effect
sex
weight
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
liver
effect
sex
burden
male &
female
%
dose
sex
10.6
timepoint
significance
%
0
n.g.
100
730
5.3
n.g.
730
20.4
n.g.
730
56.3
n.g.
0
male & f
730
10.6
male & f
50.3
male & f
250.1
male & f
additional
2.544
level
score
increased incidences of particle deposition (microspcopy) with particle accumulation in Kupffer cells
macrophages (no tissue response or damage of hepatocytes)
lung
effect
sex
inflammation
male &
female
%
dose
10.6
timepoint
LOEL
mg/kg
transient
2.544
level
score
significance
%
0
male
730
1.3
n.g.
100
0
female
730
1.3
n.g.
100
10.6
male
730
10
n.g.
769.23
10.6
female
730
15
n.g.
1153.8
50.3
male
730
11
n.g.
846.15
50.3
female
730
14
n.g.
1076.9
250.1
male
730
9.1
n.g.
700
250.1
female
730
7
n.g.
538.46
additional
28. Sep. 12
sex
LOEL
study unit
increased incidences (%) of broncho/bronchiolar pneumonia
Page 3 from 11
Titanium dioxide
effect
sex
weight
male &
female
gram
dose
sex
transient
12.072
level
91
2.4
n.g.
100
female
91
1.9
n.g.
100
male
182
2.5
n.g.
100
0
female
182
1.8
n.g.
100
0
male
365
2.6
n.g.
100
0
female
365
2.2
n.g.
100
0
male
730
3.25
n.g.
100
0
female
730
2.35
n.g.
100
10.6
male
91
2.3
none
95.83
10.6
female
91
1.7
none
89.47
10.6
male
182
2.5
none
100
10.6
female
182
2.2
none
122.22
10.6
male
365
2.7
none
103.84
10.6
female
365
2.2
none
100
10.6
male
730
3.56
none
109.53
male
0
0
score
significance
%
10.6
female
730
2.76
none
117.44
50.3
male
91
2.4
none
100
50.3
female
91
2
none
105.26
50.3
male
182
3
<0.05
120
50.3
female
182
2.6
<0.05
144.44
50.3
male
365
3.5
<0.05
134.61
50.3
female
365
3.3
<0.05
150
50.3
male
730
4.47
<0.05
137.53
50.3
female
730
3.1
<0.05
131.91
250.1
male
91
3.6
n.g.
150
250.1
female
91
2.8
n.g.
147.36
250.1
male
182
4.4
<0.05
176
250.1
female
182
4.3
<0.05
238.88
250.1
male
365
6.3
<0.05
242.3
250.1
female
365
5.7
<0.05
259.09
250.1
male
730
7.84
<0.05
241.23
250.1
female
730
7.21
<0.05
306.8
sign. dose and time-dependent increases of abs. and rel. wt.: at MD from 6-24 mo of exposure, at H
marked increase at 3 mo, sign. from 6-24 mo
effect
sex
macrophages foamy
male &
female
dose
sex
LOEL
study unit
50.3
timepoint
LOEL
mg/kg
transient
12.072
level
score
significance
%
0
male
730
18
n.g.
100
0
female
730
10
n.g.
100
10.6
male
730
27
n.g.
150
10.6
female
730
20
n.g.
200
50.3
male
730
71
n.g.
394.44
50.3
female
730
95
n.g.
950
250.1
male
730
99
n.g.
550
250.1
female
730
100
n.g.
1000
additional
28. Sep. 12
50.3
LOEL
mg/kg
timepoint
0
additional
%
LOEL
study unit
increased incidences (%) of aggregates of foamy alveolar macrophages with densely packed myelin
figures after >= 12 mo at MD and after >= 6 mo at HD indicating particle overload (Tab.3)
Page 4 from 11
Titanium dioxide
effect
sex
LOEL
study unit
female
%
dose
sex
0
male
730
0
female
730
10.6
male
730
10.6
female
50.3
male
50.3
0
n.g.
100
0
n.g.
100
94
n.g.
730
96
n.g.
730
100
n.g.
female
730
100
n.g.
250.1
male
730
100
n.g.
250.1
female
730
100
n.g.
sex
alveolar bronchiolization
male &
female
score
sex
LOEL
study unit
50.3
timepoint
LOEL
mg/kg
transient
12.072
level
score
significance
%
0
male
730
1.3
n.g.
100
0
female
730
1.3
n.g.
100
10.6
male
730
0
none
0
10.6
female
730
4
n.g.
307.69
50.3
male
730
32
n.g.
2461.5
50.3
female
730
77
n.g.
5923.1
250.1
male
730
82
n.g.
6307.7
250.1
female
730
99
n.g.
7615.4
additional
increased incidences (%) of alveolar bronchiolization
effect
sex
deposits
male &
female
dose
sex
LOEL
study unit
50.3
timepoint
LOEL
mg/kg
transient
12.072
level
score
significance
%
0
male
730
9
n.g.
100
0
female
730
2.6
n.g.
100
10.6
male
730
13
n.g.
144.44
10.6
female
730
8
n.g.
307.69
50.3
male
730
75
n.g.
833.33
50.3
female
730
72
n.g.
2769.2
250.1
male
730
97
n.g.
1077.8
250.1
female
730
96
n.g.
3692.3
additional
28. Sep. 12
level
increased incidences (%) of alveolar type II cell hyperplasia, minimal effect also seen after 6 and 12
of exposure at >= LD
effect
%
2.544
%
dose
timepoint
transient
significance
additional
%
10.6
LOEL
mg/kg
increased incidences of cholesterol granuloma and increased incidences of deposits of cholesterol,
granular or fibronous materials, cellular debris
Page 5 from 11
Titanium dioxide
effect
sex
fibrosis
male &
female
%
dose
sex
14
score
significance
%
n.g.
100
0
female
730
3.9
n.g.
100
1.6
female
730
5
none
128.2
10.6
male
730
10
none
71.42
50.3
male
730
65
n.g.
464.28
50.3
female
730
55
n.g.
1410.3
250.1
male
730
99
n.g.
707.14
female
730
99
n.g.
2538.5
increased incidences (%) of collagenized fibrosis, effect also seen at MD after 12 mo exposure, and
HD after >= 3 mo exposure
sex
macrophage damage
male &
female
dose
0
sex
male & f
LOEL
study unit
50.3
timepoint
LOEL
mg/kg
transient
12.072
level
730
score
no change
significance
50.3
male & f
730
n.a.
male & f
730
n.a.
degenerative and disintegrated foamy macrophages
effect
sex
male &
female
dose
sex
LOEL
study unit
50.3
timepoint
0
male
730
0
LOEL
mg/kg
transient
12.072
level
score
0
significance
%
n.g.
100
100
female
730
0
n.g.
10.6
male
730
0
none
10.6
female
730
0
none
50.3
male
730
51
n.g.
50.3
female
730
61
n.g.
250.1
male
730
97
n.g.
250.1
female
730
96
n.g.
additional
increased incidences (%) of alveolar proteinosis, effect also seen at MD after 12 mo exposure, and
HD after >= 3 mo exposure
effect
sex
changes in organ structure
male &
female
dose
0
250.1
additional
%
n.a.
250.1
additional
28. Sep. 12
level
730
effect
n.a.
timepoint
transient
12.072
male
250.1
%
50.3
LOEL
mg/kg
0
additional
n.a.
LOEL
study unit
sex
LOEL
study unit
250.1
timepoint
male & f
730
male & f
730
LOEL
mg/kg
transient
60.024
level
score
no change
significance
%
n.a.
n.a.
alveoli lined with ciliated columnar cells dilated and coalesced to form large multiple cystic spaces w
mucinous secretions and dust cells
Page 6 from 11
Titanium dioxide
effect
sex
male &
female
%
dose
sex
0
male
730
0
60.024
timepoint
level
score
2.5
significance
%
n.g.
100
730
0
n.g.
100
male
730
1.4
none
56
10.6
female
730
0
n.g.
50.3
male
730
1.4
none
50.3
female
730
0
n.g.
250.1
male
730
15.6
n.g.
250.1
female
730
17.6
n.g.
clearance
male &
female
dose
sex
LOEL
study unit
10.6
timepoint
LOEL
mg/kg
2.544
level
score
significance
male & f
91
no change
n.a.
0
male & f
182
no change
n.a.
0
male & f
365
no change
n.a.
0
male & f
730
no change
10.6
male & f
91
10.6
male & f
182
n.a.
10.6
male & f
365
n.a.
10.6
male & f
730
n.a.
50.3
male & f
91
n.a.
50.3
male & f
182
n.a.
50.3
male & f
365
n.a.
50.3
male & f
730
n.a.
250.1
male & f
91
n.a.
250.1
male & f
182
n.a.
250.1
male & f
365
n.a.
male & f
730
n.a.
250.1
sex
collagen
male &
female
0
n.a.
n.a.
sex
male & f
LOEL
study unit
50.3
timepoint
730
LOEL
mg/kg
transient
12.072
level
score
no change
significance
%
n.a.
50.3
male & f
730
n.a.
250.1
male & f
730
n.a.
additional
%
particle-laden macrophages in alveoli seen at all doses after 3 mo of exposure, effect with dose- and
time-response-relationship
effect
dose
624
transient
0
additional
56
increased incidence (%) at HD
sex
28. Sep. 12
transient
female
effect
n.a.
250.1
LOEL
mg/kg
10.6
additional
n.a.
LOEL
study unit
minute collagen fiber deposition in alveolar walls at MD and HD, sign. collagen fiber deposition in
cholesterol granulomas at HD at termination of study; effect not seen at LD
Page 7 from 11
Titanium dioxide
effect
sex
burden
male &
female
mg/organ
dose
sex
LOEL
study unit
10.6
LOEL
mg/kg
transient
2.544
timepoint
level
91
0.001
n.g.
100
female
91
0.001
n.g.
100
male
182
0.001
n.g.
100
0
female
182
0.001
n.g.
100
0
male
365
0.001
n.g.
100
0
female
365
0.001
n.g.
100
0
male
730
0.001
n.g.
100
0
female
730
0.001
n.g.
100
10.1
female
365
8.7
n.g.
870000
10.6
male
91
2.5
n.g.
250000
10.6
female
91
2.8
n.g.
280000
10.6
male
182
4.8
n.g.
480000
10.6
female
182
4.4
n.g.
440000
10.6
male
365
10.1
n.g.
1E+06
10.6
male
730
20.7
n.g.
2E+06
10.6
female
730
32.3
n.g.
3E+06
50.3
male
91
21.7
n.g.
2E+06
50.3
female
91
16.6
n.g.
2E+06
50.3
male
182
57.3
n.g.
6E+06
50.3
female
182
54
n.g.
5E+06
50.3
male
365
75.6
n.g.
8E+06
50.3
female
365
59.7
n.g.
6E+06
50.3
male
730
118.3
n.g.
1E+07
50.3
female
730
130
n.g.
1E+07
150.1
female
365
381.5
n.g.
4E+07
250.1
male
91
180.8
n.g.
2E+07
250.1
female
91
136.8
n.g.
1E+07
250.1
male
182
275.3
n.g.
3E+07
250.1
female
182
238.6
n.g.
2E+07
250.1
male
365
361.7
n.g.
4E+07
250.1
male
730
784.8
n.g.
8E+07
250.1
female
730
545.8
n.g.
5E+07
0
male
0
0
additional
effect
score
significance
%
dose- and time-dependent increase of lung burden (mg/lung, detection limit < 0,002 mg/g dry lung);
particle overload at HD based on impaired lung clearance after >= 12 mo exposure
sex
LOEL
study unit
LOEL
mg/kg
transient
tumor
%
additional
28. Sep. 12
no effect: single case of large cell anaplastic carcinoma in 1/71 LD m, effect not seen at higher dose
in f; effect probably not treatment-dependent
Page 8 from 11
Titanium dioxide
effect
sex
male &
female
%
LOEL
study unit
250.1
timepoint
LOEL
mg/kg
transient
60.024
dose
sex
significance
%
0
male
730
level
0
score
n.g.
100
0
100
female
730
0
n.g.
10.6
male
730
0
n.g.
10.6
female
730
1.3
n.g.
50.3
male
730
0
n.g.
50.3
female
730
0
n.g.
250.1
male
730
1.3
n.g.
250.1
female
730
17.6
n.g.
additional
increased incidence (%) in HD f (13/74), single cases in HD m (1/75) and LD f (1/75)
lymph node
effect
sex
burden
male &
female
%
dose
sex
LOEL
study unit
50.3
timepoint
%
n.g.
100
730
0
none
730
4.5
n.g.
730
61.2
n.g.
730
10.6
male & f
50.3
male & f
250.1
male & f
burden
male &
female
10
sex
timepoint
score
LOEL
study unit
LOEL
mg/kg
transient
2.4
significance
%
0
male & f
730
0
n.g.
100
10.6
male & f
730
31.5
n.g.
50.3
male & f
730
78
n.g.
250.1
male & f
730
93.3
n.g.
additional
sex
burden
male &
female
dose
level
score
Increased incidence of particle deposition (microspcopy) in cervical lymph nodes with particle-laden
accumulation
effect
%
level
Increased incidenceof particle deposition (microspcopy) in mesenteric lymph nodes with particle-lad
cell accumulation: slight increase at MD and distinct effect at HD
sex
dose
12.072
significance
male & f
effect
%
transient
0
0
additional
LOEL
mg/kg
10.6
timepoint
LOEL
mg/kg
transient
2.544
significance
%
0
male & f
730
0
n.g.
100
10.6
male & f
730
87.2
n.g.
50.3
male & f
730
96.6
n.g.
250.1
male & f
730
100
n.g.
additional
sex
LOEL
study unit
level
score
Increased incidence of particle deposition (microspcopy) in tracheobronchial lymph nodes with partic
laden cell accumulation
nose
28. Sep. 12
Page 9 from 11
Titanium dioxide
effect
sex
metaplasia
male &
female
%
dose
sex
10.6
LOEL
mg/kg
male
730
0
transient
2.544
timepoint
0
level
score
significance
%
10
n.g.
100
female
730
9
n.g.
100
10.6
male
730
37
n.g.
370
10.6
female
730
19
n.g.
211.11
50.3
male
730
27
n.g.
270
50.3
female
730
28
n.g.
311.11
250.1
male
730
58
n.g.
580
female
730
55
n.g.
611.11
250.1
additional
increased incidences of anterior squamous metaplasia (%); no increase of posterior squamous
metaplasia
effect
sex
rhinitis
male &
female
%
LOEL
study unit
dose
sex
LOEL
study unit
10.6
LOEL
mg/kg
transient
2.544
timepoint
level
score
significance
%
0
male
730
32
n.g.
100
0
female
730
24
n.g.
100
10.6
male
730
80
n.g.
250
10.6
female
730
49
n.g.
204.16
50.3
male
730
66
n.g.
206.25
50.3
female
730
46
n.g.
191.66
250.1
male
730
92
n.g.
287.5
250.1
female
730
86
n.g.
358.33
additional
increased incidences of anterior rhinitis (%), no clear dose-response; incidences of posterior rhinitis
were not increased in m, increases in f only at LD and HD
pleura
effect
sex
inflammation
male &
female
%
LOEL
study unit
10.6
LOEL
mg/kg
transient
2.544
dose
sex
significance
%
0
male
730
5.1
n.g.
100
0
timepoint
level
score
female
730
2.6
n.g.
100
10.6
male
730
10
n.g.
196.07
10.6
female
730
9
n.g.
346.15
50.3
male
730
37
n.g.
725.49
50.3
female
730
35
n.g.
1346.2
89
female
730
89
n.g.
3423.1
male
730
71
n.g.
1392.2
250.1
additional
increased incidences of pleuritis
spleen
28. Sep. 12
Page 10 from
Titanium dioxide
effect
sex
burden
male &
female
%
dose
10.6
timepoint
LOEL
mg/kg
transient
2.544
significance
%
0
male & f
730
0
n.g.
100
10.6
male & f
730
10.6
n.g.
50.3
male & f
730
19.3
n.g.
250.1
male & f
730
67.8
n.g.
additional
sex
LOEL
study unit
level
score
increased particle deposition (microspcopy) with particle accumulation mostly in white pulp with
occasional aggregates of foamy macrophages (no cellular changes in spleen)
trachea
effect
sex
tracheitis
male &
female
%
dose
10.6
timepoint
LOEL
mg/kg
transient
2.544
significance
%
0
male
730
3
n.g.
100
level
score
0
female
730
1.3
n.g.
100
10.6
male
730
76
n.g.
2533.3
10.6
female
730
46
n.g.
3538.5
50.3
male
730
72
n.g.
2400
50.3
female
730
50
n.g.
3846.2
250.1
male
730
79
n.g.
2633.3
250.1
female
730
43
n.g.
3307.7
additional
28. Sep. 12
sex
LOEL
study unit
sign. increase of incidences (%) to similar levels in all dosed m or f, m more sensitive (higher
incidences) than f
Page 11 from 11
Silver
g/mol
Study Data
study pk 4114
object
Primary particle
synonym
Silver nanoparticles (self-made)
size
diameter in nm
mean
18.48
SD
1.45
min
6
max
55
medium
TEM (count median diameter)
determ. method
distribution type
no data
surface property
spherical
cristal structure
shape
specific surface in
m²/g
solubility in mg/l
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
length in nm
reference
Self made: freshly prepared nanoparticles by means of evaporation/condensation with
ceramic heater; no-aggregated particles (same technique as for 28 d study of Ji et al., 2007)
Sung et al., 2008, 2009
producer
Self made: Korea Environment & Merchandise Testing Institute, Korea
object
size
Primary particle
diameter in nm
length in nm
mean
SD
min
max
medium
distribution type
determ. method
no data
surface property
cristal structure
shape
specific surface in
m²/g
solubility in mg/l
specific volume in
m³/g
at in °C
particle density in
g/cm³
additional
reference
median
nm
GSD
min
01. Oct. 12
distibution type
bulk density
nm
isoelectric point
monodispers
mg/ml
in
Page 1 from 16
Silver
max
nm
medium
determ. method
differential mobility
analyzer and
ultrafine condensation
particle counter
sample treatment
zeta potential
mV
in
peak SPR
nm
in
conductivity
µS/cm in
solubility
mg/L
in
at
ºC
applic. medium
dispersant
geometric mean diameter and GSD in aerosol (LD; MD; HD): 18,12 +-1,31 nm; 18,33 +- 1,12
nm; 18,93 +- 1,59 nm
additional
Study Design
species
rat
strain
Sprague-Dawley
sex
male & female
animal/group
10
route
whole-body
age of animal
8w
exposure in h/d
6
exposure in d/w
5
study dur. in d
91
postexp. dur. in d
0
purity
no. of instillation
frequency
exposure
(additional)
dose /
concentration
whole-body 1,3 qm exposure chamber; analytical conc.: 48,94+-0,47; 133,19+-1,05; 514,78+3,74 µg/m³; particle conc.: 66400; 1430000; 2850000 particles/cm³; surface area: 1,08; 2,37;
6,61 x109 nm²/cm³; bw m 253 g, f 162 g
0
0.048
0.133
0.515
Unit
mg/m³
1 dose study
reliability
A
confidential
Lung function weekly by whole-body plethysmography 40 min after termination of daily exposure;
other endpoints on d 90; Study according to OECD guideline 413 (1995)
Ag burden by AAS after tissue digestion
additional
Reference
author
volume
Kim JS, et al.
2
source
year
Safe Health Work
2011
institution
Toxicological Research Center, Hoseo University, Korea
author
Sung J, et al.
source
volume
108
year
2009
institution
Korea Environment & Merchandise Testing Institute, Korea
author
Sung J, et al.
source
volume
20
year
2008
institution
Korea Environment & Merchandise Testing Institute, Korea
page
34-38
page
452-461
page
567-574
Scope
animal/group
organ
necropsy
guideline
BALF
4
total protein
PMN
albumin
lymphocytes
additional
01. Oct. 12
14x3 ml PBS, total lung
Page 2 from 16
Silver
clinical symptoms
additional
body weight
10
including food consumption and signs of irritancy
10
lung
6
additional
organ weight for left and right lung lobe, no lavage (N=6); lung burden (total lung,
no lavage, N=4-5); Histopatho: H&E stain; lung function parameters: tidal and
minute volume, respiratory frequency, inspiration and expiration time, peak
inspiration and expiration flow
10
additional
organ weight for left and right testis
10
testes
kidney
additional
spleen
weight of left and right kidney; organ burden (N=4-5
10
liver
10
additional
adrenal gland
additional
heart
organ burden (N=3-5)
10
organ weight for left and right adrenal
10
thymus
10
brain
10
additional
organ burden (2-5 m and 4-5 f per dose)
10
additional
weight and burden of olfactory bulb (N=3-5)
10
additional
organ weight for left and right ovary
10
nose
ovary
haematology
hematocrit
haemoglobin
erythrocyte aggregation
partial thromboplastin time
Prothrombin time
Monocytes
basophils
eosinophils
Lymphocytes
Red cell distribution width
Neutrophils
Reticulocytes
Total leukocyte count
Platelet count
Mean corpuscular haemoglobin conc
Mean corpuscular volume
Mean corpuscular haemoglobin
Platelet volume
differential leukocyte count
clinical chemistry
10
thyroid gland
10
bladder
10
uterus
10
epididymis
10
seminal vesicle
10
trachea
10
01. Oct. 12
erythrocyte count
Page 3 from 16
Silver
oesophagus
10
tongue
10
prostate
10
pancreas
10
urine analysis
5
Total protein
ß-NAG
bone marrow
additional
femurs: in vivo micronucleus test (OECD GL 474)
Effect data
adrenal gland
effect
sex
histopathology
male &
female
additional
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
effect
sex
weight
male &
female
additional
LOEL
study unit
no effect
BALF
effect
sex
albumin
female
µg/ml
dose
sex
0.515
0.11705
timepoint
level
8.3
0
male
91
0
score
significance
%
n.g.
100
female
91
9.3
n.g.
100
0.048
male
91
16.5
none
198.79
0.048
female
91
11.3
none
121.5
0.133
male
91
11.8
none
142.16
0.133
female
91
11.8
none
126.88
0.515
male
91
1.8
none
21.68
female
91
37.75
<0.05
405.91
0.515
additional
sign. increase in HD f (mean with high SD: 37,75 +- 20,1), however, in HD m a decrease was seen t
was sign. compared to LD and MD values
effect
sex
lymphocytes total
male &
female
LOEL
study unit
LOEL
mg/kg
transient
x10E6
additional
01. Oct. 12
no effect: number of lymphocytes
Page 4 from 16
Silver
effect
sex
PMN total
male &
female
LOEL
study unit
LOEL
mg/kg
transient
LOEL
mg/kg
transient
LOEL
mg/kg
transient
LOEL
mg/kg
transient
x10E6
additional
no effect: number of PMN
effect
sex
male &
female
LOEL
study unit
x10E6
additional
no effect: number of AM
effect
sex
total cells
male &
female
LOEL
study unit
x10E6
additional
effect
no effect: total cells
sex
total protein
female
µg/ml
0.515
dose
sex
timepoint
0
male
91
0
female
91
0.048
male
0.048
0.11705
significance
%
13.3
n.g.
100
13.9
n.g.
100
91
16.5
none
124.06
female
91
10.4
none
74.82
0.133
male
91
15.1
none
113.53
0.133
female
91
10.9
none
78.41
0.515
male
91
13.8
none
103.75
0.515
female
91
20.33
<0.05
146.25
additional
level
sex
female
LOEL
study unit
0.515
timepoint
LOEL
mg/kg
transient
0.11705
dose
sex
0
male
91
55.3
level
0
female
91
57.3
0.048
male
91
73.4
0.048
female
91
41.8
0.133
male
91
69.1
0.133
female
91
41.8
0.515
male
91
73
0.515
female
91
116.7
additional
score
sign. increase in HD f; no increase in m
effect
units/liter (U/l)
LOEL
study unit
score
sign. increase of LDH activity in HD f (2fold); no increase in m
bladder
01. Oct. 12
Page 5 from 16
Silver
effect
sex
histopathology
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
body weight
effect
sex
weight
male &
female
additional
no effect
bone
effect
sex
histopathology
male &
female
additional
no effect
bone marrow
effect
sex
micronuclei
male &
female
additional
no effect: no increased frequencies of micronucleated polychromatic erythrocytes
brain
effect
sex
burden
male &
female
µg/g wet organ
dose
sex
0.133
LOEL
mg/kg
transient
0.03023
timepoint
level
0
male
91
0.00112
0
score
significance
%
n.g.
100
female
91
0.00066
n.g.
100
0.048
male
91
0.00345
none
308.03
0.048
female
91
0.00409
none
619.69
0.133
male
91
0.00789
<0.01
704.46
0.133
female
91
0.0102
<0.01
1545.5
0.515
male
91
0.0186
<0.01
1660.7
0.515
female
91
0.02
<0.01
3030.3
additional
sign. increase of brain burden (Tab.7, 8)
effect
sex
histopathology
male &
female
additional
01. Oct. 12
LOEL
study unit
LOEL
study unit
LOEL
mg/kg
transient
no effect
Page 6 from 16
Silver
effect
sex
weight
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
clinical chemistry
effect
sex
parameters acc. to picklist
male &
female
additional
no effect
clinical symptoms
effect
sex
food consumption
male &
female
additional
no effect: including food consumption
epididymis
effect
sex
histopathology
male
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
haematology
effect
sex
erythrocyte aggregation
%
female
dose
sex
0.515
timepoint
0.11705
level
score
significance
%
0
female
91
8
n.g.
100
0.048
female
91
4
none
50
0.133
female
91
19
none
237.5
0.515
female
91
28
<0.01
350
additional
sign. increase of % aggregated erythrocytes in HD f
heart
effect
sex
weight
male &
female
additional
01. Oct. 12
LOEL
study unit
LOEL
mg/kg
transient
no effect
Page 7 from 16
Silver
effect
sex
histopathology
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
kidney
effect
sex
histopathology
male &
female
additional
no effect
effect
sex
burden
male &
female
µg/g wet organ
dose
sex
0.133
0.03023
timepoint
level
score
significance
%
0
male
91
0.00085
n.g.
100
0
female
91
0.00094
n.g.
100
0.048
male
91
0.00163
none
191.76
0.048
female
91
0.00261
none
277.65
0.133
male
91
0.00358
<0.01
421.17
0.133
female
91
0.0118
none
1255.3
0.515
male
91
0.00949
<0.01
1116.5
0.515
female
91
0.0377
<0.01
4010.6
additional
m more sensitive, sign. increase of kidney burden in f at HD only; however, at MD and HD absolute
kidney burdens of f were 3-4 fold higher than in m (Tab.7, 8)
effect
sex
weight
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
liver
effect
sex
burden
male &
female
µg/g wet organ
dose
0.11705
timepoint
level
0
male
91
0.0007
0
score
significance
%
n.g.
100
female
91
0.0009
n.g.
100
0.048
male
91
0.00352
none
502.85
0.048
female
91
0.00455
none
505.55
0.133
male
91
0.0138
none
1971.4
0.133
female
91
0.0121
none
1344.4
0.515
male
91
0.133
<0.01
19000
female
91
0.071
<0.01
7888.9
0.515
additional
01. Oct. 12
sex
0.515
sign. increase at HD only (Tab.7, 8)
Page 8 from 16
Silver
effect
sex
hyperplasia
male &
female
%
dose
level
score
significance
%
0
minimal
n.g.
100
0
female
91
30
minimal
n.g.
100
0.048
male
91
0
minimal
none
0
0.048
female
91
20
minimal
none
66.66
0.133
male
91
10
minimal
none
33.33
0.133
female
91
40
minimal
none
133.33
0.515
male
91
44.4
minimal
<0.05
148
0.515
female
91
10
medium
<0.05
33.33
female
91
80
minimal
<0.05
266.66
sign. increase of incidence of minimal bile-duct hyperplasia at HD
sex
necrosis
female
dose
sex
0.515
timepoint
LOEL
mg/kg
transient
0.11705
level
score
significance
%
100
0
female
91
0
n.g.
female
91
0
none
0.133
female
91
0
none
0.515
female
91
30
<0.05
sign. increase of incidence of single-cell hepatocellular necrosis in HD f (effect not seen in m); multif
hyperplasia of minimal grade seen in HD m (11.1%) and control f (20%) and of moderate grade in 1
HD f, effect not seen in other groups
effect
sex
fibrosis
female
dose
0
0.515
additional
sex
0.515
effect
female
91
10
sex
LOEL
study unit
0.515
timepoint
LOEL
mg/kg
score
medium
significance
%
n.g.
100
none
transient
0.11705
level
female
91
0
female
91
10
score
medium
significance
%
n.g.
100
none
single HD f with mild pigment accumulation together with moderate bile-duct hyperplasia, minimal si
cell hepatocyte necrosis, moderate centrilobular fibrosis and moderate multifocal necrosis
sex
vacuolization
level
single HD f with moderate centrilobular fibrosis together with moderate bile-duct hyperplasia, minima
single-cell hepatocyte necrosis, mild pigment accumulation and moderat multifocal necrosis
female
additional
timepoint
transient
0.11705
0
pigmentation
0
0.515
LOEL
mg/kg
91
sex
dose
LOEL
study unit
female
effect
male
LOEL
study unit
0.515
transient
0.11705
sex
0
male
91
0
female
91
0.048
female
91
10
minimal
none
male
91
11.1
minimal
none
0.515
timepoint
LOEL
mg/kg
dose
additional
01. Oct. 12
LOEL
study unit
0.048
additional
%
0.11705
91
effect
%
timepoint
transient
male
0.515
%
0.515
LOEL
mg/kg
0
additional
%
sex
LOEL
study unit
level
score
significance
%
0
n.g.
100
0
n.g.
100
single HD m with hepatocellular vacuolation; effect also seen in single LD f but not at other doses, e
in f no considered to be substance-dependent
Page 9 from 16
Silver
effect
sex
mineralization
%
male
0.515
LOEL
mg/kg
sex
0
male
91
0
0.515
male
91
11.1
timepoint
level
sex
granuloma
male &
female
sex
LOEL
study unit
significance
%
n.g.
100
minimal
none
timepoint
LOEL
mg/kg
transient
level
score
significance
%
n.g.
100
minimal
none
0
female
91
0
0.048
female
91
20
additional
score
single HD m with minimal portal mineralization together with minimal bile-duct hyperplasia
effect
dose
transient
0.11705
dose
additional
%
LOEL
study unit
no effect: 2 LD f with minimal multifocal granuloma, effect not seen at higher doses and not conside
to be substance-dependent
effect
sex
weight
male &
female
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
gram
additional
no effect
lung
effect
sex
thickening
male &
female
%
dose
0
0.515
additional
sex
0.515
0.11705
timepoint
male & f
91
male & f
91
level
score
no change
significance
%
n.a.
n.a.
sign. increased incidence of alveolar wall thickening (Tab.9, 10; see inflammation)
effect
sex
weight
male &
female
LOEL
study unit
LOEL
mg/kg
transient
gram
additional
01. Oct. 12
no effect: total wt. (no lavage)
Page 10 from 16
Silver
effect
sex
inflammation
male &
female
%
dose
sex
0.11705
level
score
significance
%
91
20
minimal
n.g.
100
0
female
91
30
minimal
n.g.
100
0.048
male
91
30
minimal
n.g.
150
0.048
female
91
20
minimal
n.g.
66.66
0.133
male
91
20
minimal
n.g.
100
0.133
female
91
0
minimal
n.g.
0
0.515
male
91
89
minimal
n.g.
350
0.515
female
91
80
minimal
n.g.
266.66
increased incidence of minimal chronic alveolar inflammation including alveolitis, granulomatous
lesions, and alveolar wall thickening (Tab.9, 10)
sex
infiltration
male &
female
dose
sex
LOEL
study unit
0.133
timepoint
0
male
91
0
LOEL
mg/kg
transient
0.03023
level
score
significance
%
30
minimal
n.g.
100
female
91
0
minimal
n.g.
100
0.048
male
91
40
minimal
n.g.
133.33
0.048
female
91
0
minimal
n.g.
0.133
male
91
60
minimal
n.g.
0.133
female
91
10
minimal
n.g.
0.515
male
91
78
minimal
n.g.
0.515
female
91
70
minimal
n.g.
additional
sex
tidal volume
male &
female
dose
200
266.66
Minimal perivascular infiltration of mixed cells: m more sensitive, LOEL in f at 0,515 mg/m3 (Tab.9, 1
effect
sex
LOEL
study unit
0.048
timepoint
0
male
21
0
female
0
male
0.048
LOEL
mg/kg
transient
0.01091
level
score
significance
%
0.265
n.g.
70
0.24
n.g.
100
91
0.305
n.g.
100
male
21
0.225
<0.01
0.97
0.048
female
70
0.22
none
91.66
0.048
male
91
0.275
<0.01
90.16
0.133
male
21
0.21
<0.01
0.91
0.133
female
70
0.21
<0.01
87.5
0.133
male
91
0.265
<0.01
86.88
0.515
male
21
0.195
<0.01
0.84
0.515
female
70
0.195
<0.01
81.25
0.515
male
91
0.26
<0.01
85.24
additional
01. Oct. 12
timepoint
transient
male
effect
ml
0.515
LOEL
mg/kg
0
additional
%
LOEL
study unit
dose-dependent decrease of tidal volume: sign. at single tps, effect more pronounced in m (Fig.3A,
Page 11 from 16
Silver
effect
sex
burden
male &
female
µg/g wet organ
dose
sex
timepoint
level
91
0.00077
0
score
significance
%
n.g.
100
female
91
0.00101
n.g.
100
0.048
male
91
0.614
none
79740
0.048
female
91
0.296
none
29307
0.133
male
91
5.45
<0.01
707792
0.133
female
91
4.241
<0.01
419901
0.515
male
91
14.65
<0.01
2E+06
female
91
20.59
<0.01
2E+06
sign. dose-dependent increase of total lung burden (no lavage): effect at LD not statistically sign.
(Tab.7,8)
effect
sex
peak inspiration flow
male &
female
dose
sex
LOEL
study unit
0.048
LOEL
mg/kg
timepoint
level
male
14
1.25
0
female
14
0.048
male
14
0.048
female
14
0.133
male
14
0.133
female
14
0.515
male
14
0.515
female
14
score
significance
n.g.
no change
0.9
%
100
n.a.
72
<0.01
n.a.
1.08
<0.01
86.4
n.a.
0.8
<0.01
64
n.a.
dose-dependent decrease of peak inspiration flows in m & f of all groups (partly sign.) (Fig.3C, f not
shown)
effect
sex
minute volume
male &
female
dose
transient
0.01091
0
additional
sex
LOEL
study unit
0.048
LOEL
mg/kg
transient
0.01091
timepoint
level
0
male
14
27.5
n.g.
100
0
female
28
23
n.g.
100
0
male
49
28
n.g.
100
0.048
male
14
18.5
<0.05
67.27
0.048
female
28
18.5
<0.01
80.43
0.048
male
49
24
<0.01
85.71
0.133
male
14
24
<0.05
87.27
0.133
female
28
18
<0.01
78.26
0.133
male
49
23
<0.01
82.14
0.515
male
14
18
<0.05
65.45
0.515
female
28
15.7
<0.01
68.26
0.515
male
49
20
<0.01
71.42
additional
01. Oct. 12
transient
0.03023
male
0.515
ml/min
0.133
LOEL
mg/kg
0
additional
ml/s
LOEL
study unit
score
significance
%
dose-dependent decrease of minute volume in m & f of all groups (partly sign.) (Fig.3B, 4B)
Page 12 from 16
Silver
effect
sex
granuloma
male &
female
%
dose
0
male & f
0.515
LOEL
mg/kg
transient
0.11705
timepoint
level
score
no change
n.a.
n.a.
male & f
91
no change
0.133
male & f
91
no change
0.515
male & f
91
effect
male
dose
%
n.a.
n.a.
sign. increased incidence of granulomatous lesions (Tab.9, 10; see inflammation)
sex
significance
91
0.048
additional
%
sex
LOEL
study unit
sex
LOEL
study unit
0.515
LOEL
mg/kg
transient
0.11705
timepoint
level
score
significance
%
0
male
91
30
minimal
n.g.
100
0
female
91
70
minimal
n.g.
100
0.048
male
91
50
minimal
n.g.
166.66
0.048
female
91
40
minimal
n.g.
57.14
0.113
male
91
50
minimal
n.g.
166.66
0.133
female
91
40
minimal
n.g.
57.14
0.515
male
91
89
minimal
n.g.
266.66
0.515
female
91
60
minimal
n.g.
85.71
additional
minimal accumulation of alveolar macrophages: increased incidence in HD m; no increase in f due t
high background incidence (Tab.9, 10)
nose
effect
sex
burden
male &
female
µg/g wet organ
dose
sex
0.515
LOEL
mg/kg
transient
0.11705
timepoint
level
score
significance
%
0
male
91
0.00051
n.g.
100
0
female
91
0.00226
n.g.
100
0.048
male
91
0.00644
none
1262.7
0.048
female
91
0.00743
none
328.76
0.133
male
91
0.0171
none
3352.9
0.133
female
91
0.0138
none
610.61
0.515
male
91
0.0305
<0.01
5980.4
0.515
female
91
0.00328
<0.01
145.13
additional
sign. dose-dependent increase in olfactory bulb (Tab.7, 8)
effect
sex
weight
male &
female
additional
sex
histopathology
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
mg/kg
transient
no effect: wt. of olfactory bulb
effect
01. Oct. 12
LOEL
study unit
LOEL
study unit
no effect
Page 13 from 16
Silver
oesophagus
effect
sex
histopathology
male &
female
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
ovary
effect
sex
weight
female
additional
no effect
effect
sex
histopathology
female
additional
no effect
pancreas
effect
sex
histopathology
male &
female
additional
no effect
prostate
effect
sex
histopathology
male
additional
no effect
seminal vesicle
effect
sex
histopathology
male
additional
no effect
testes
effect
sex
weight
male
additional
01. Oct. 12
no effect
Page 14 from 16
Silver
effect
sex
histopathology
male
additional
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
LOEL
study unit
LOEL
mg/kg
transient
no effect
thymus
effect
sex
histopathology
male &
female
additional
no effect
effect
sex
weight
male &
female
additional
no effect
thyroid gland
effect
sex
histopathology
male &
female
additional
no effect
trachea
effect
sex
histopathology
male &
female
additional
no effect
urine analysis
effect
sex
protein
g/g creatinine
male
dose
sex
0.515
0.11705
timepoint
level
score
significance
%
0
male
91
1.89
n.g.
100
0
female
91
3.88
n.g.
100
0.048
male
91
1.58
none
83.59
0.048
female
91
0.9
none
23.19
0.133
male
91
2.39
none
126.45
0.133
female
91
2.93
none
75.51
0.515
male
91
2.57
<0.05
135.97
0.515
female
91
2.26
none
58.24
additional
sign. increase of protein in urine in HD m, effect not seen in f due to high mean control value with hig
SD
uterus
01. Oct. 12
Page 15 from 16
Silver
effect
sex
histopathology
female
additional
01. Oct. 12
LOEL
study unit
LOEL
mg/kg
transient
no effect
Page 16 from 16

Carcinogenicity and Mutagenicity of Nanoparticles - Biozid

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